Methods and Kits for Methylation Detection

ABSTRACT

Ligation-based methods and kits are disclosed for determining the degree of methylation of one or more target nucleotides. In certain embodiments, the methylation status of one or more target nucleotides is determined by generating misligation products. In certain embodiments, at least one target nucleotide is amplified prior to the ligation reaction. In certain embodiments, at least one ligation product, at least one ligation product surrogate, at least one misligation product, at least one misligation product surrogate, or combinations thereof are amplified. In certain embodiments, one or more ligation probes comprise at least one nucleotide analog, at least one Modification, at least one mismatched nucleotide, or combinations thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/119,985,filed May 2, 2005, which claims a priority benefit under 35 U.S.C.§119(e) from application No. 60/567,396, filed Apr. 30, 2004, which areall incorporated herein by reference.

FIELD

The present teachings generally relate to methods and kits fordetermining the methylation state of at least one nucleotide in nucleicacid sequences of interest. More specifically, the teachings relate toligation-based methods and kits for determining the degree ofmethylation of target nucleotides.

BACKGROUND

The methylation of cytosine residues in DNA is an important epigeneticalteration in eukaryotes. In humans and other mammals methylcytosine isfound almost exclusively in cytosine-guanine (CpG) dinucleotides. DNAmethylation plays an important role in gene regulation and changes inmethylation patterns are reportedly involved in human cancers andcertain human diseases. Among the earliest and most common geneticalterations observed in human malignancies is the aberrant methylationof CpG islands, causing the over-expression or silencing of many genes.Subsequently, there is great interest in using DNA methylation markersas diagnostic indicators for early detection, risk assessment,therapeutic evaluation, recurrence monitoring, and the like. (Seegenerally, Laird, Nature Reviews, 3:253-266, 2003; Fraga et al.,BioTechniques 33:632-49, 2002; Adorjan et al., Nucleic Acids Res.30(5):e21, 2002; and Colella et al., BioTechniques, 35(1):146-150,2003). There is also great scientific interest in DNA methylation forstudying and modifying gene regulation, among other things.

SUMMARY

Methods and kits are provided for determining the degree of methylationof specific target nucleotides, generally but not exclusively cytosineresidues, in target nucleic acid sequences, typically genomic DNA(gDNA). The methods and kits generally employ at least one probe setcomprising at least one first probe and at least one second probe that,under appropriate conditions, are ligated together using at least oneligation agent, to form at least one (mis)ligation product. By detectingat least some of these ligation products or their surrogates (e.g.,digested ligation products, amplified ligation products, digestedamplified ligation products, reporter probes or at least portions ofreporter probes, and the like), one can determine the degree ofmethylation for the corresponding target nucleotide(s).

In certain embodiments, the presence of a methyl group on at least onetarget nucleotide affects the ability of at least one ligation agent togenerate one or more ligation product species. By comparing theexperimentally determined ligation rate for a given ligation agent andone or more probe sets with the control ligation rates (typically usingthe same probe set with control target nucleic acid sequences of knownmethylation status and the same ligation agent), the degree ofmethylation of at least one target nucleotide species can be determined.In certain embodiments, the presence of a methyl group on at least onetarget nucleotide affects the ability of at least one ligation agent togenerate one or more misligation product species. That is, at least onenucleotide in the target-specific portion of at least one probe in aprobe set is not fully complementary with the corresponding bindingregion of the target nucleic acid sequence, for example but not limitedto the target nucleotide, yet the two corresponding probes arenevertheless joined, i.e., misligated by a ligation agent. By comparingthe experimental misligation rate with the control misligation rates orappropriate standard curves, the degree of methylation of at least onetarget nucleotide can be determined. Control ligation/misligation ratescan be pre-determined, analyzed in one or more parallel reaction, ordetermined subsequently. In certain embodiments, ligation and/ormisligation occurs when the target nucleotide is not methylated but doesnot occur or occurs at a lower rate than when the target nucleotide ismethylated. In certain embodiments, ligation/misligation is enhancedwhen the target nucleotide is methylated relative to theligation/misligation rate when the target nucleotide is not methylated.

In certain embodiments, the (mis)ligation rate is affected by thepresence of one or more Modifications in at least one probe of at leastone probe set. In certain embodiments, the 3′-end of the hybridizedupstream probe, the 5′-end of the hybridized downstream probe, or both(i.e., the ligation site), is directly opposite one or more targetnucleotide. In certain embodiments, at least one ligation site isupstream from or downstream from one or more target nucleotide beinginterrogated. In certain embodiments, at least two probe sets forinterrogating the same target nucleotide have different ligation sites.These at least two probe sets may, but need not be, competed againsteach other in an assay.

In certain embodiments, (mis)ligation products are amplified using atleast one polymerase to generate amplified (mis)ligation products. Incertain embodiments, at least one amplified (mis)ligation product orother (mis)ligation product surrogate is amplified using an amplifyingmeans such as at least one polymerase. In certain embodiments, at leastone (mis)ligation product, at least one amplified (mis)ligation product,or at least one (mis)ligation product and at least one amplified(mis)ligation product, is combined with at least one digestion means,such as an enzyme (including but not limited to at least oneendonuclease, at least one exonuclease, at least one restriction enzyme,or combinations thereof) or chemical digesting means, to generate atleast one digested (mis)ligation product, at least one digestedamplified (mis)ligation product, or at least one digested (mis)ligationproduct and at least one digested amplified (mis)ligation product. Atleast one (mis)ligation product, at least one (mis)ligation productsurrogate, or combinations thereof, are detected and the degree ofmethylation of the corresponding target nucleotides are determined. Incertain embodiments, at least one (mis)ligation product, at least one(mis)ligation product surrogate, or combinations thereof, comprises atleast one reporter group, at least one mobility modifier, at least onehybridization tag, at least one reporter probe-binding portion, at leastone affinity tag, or combinations thereof that, among other things,facilitate determining the degree of target nucleotide methylation.Competitive ligation reactions, wherein at least two competing ligationprobes compete with each other to hybridize with the same orsubstantially the same target nucleic acid sequence comprising at leastone target nucleotide are within the scope of the teachings herein. Incertain embodiments, determining the degree of methylation of at leastone target nucleotide comprises comparing the ratio of (mis)ligationproducts, (mis)ligation product surrogates, or combinations thereof, forexample but not limited to visual, automated, or semi-automatedcomparison of peak heights, peak areas, signal intensity, and the like.In certain embodiments, determining comprises using one or more computeralgorithm.

Pretreatment of the target nucleic acid sequences with sodium bisulfiteor other chemical modifying agent is not required (and generally notpreferred), nor is enzymatic cleavage with methylation sensitiverestriction endonuclease pairs, such as the isoschisomers HpaII/MspI,EcoRII/BstNI, or the like (see REBASE database at “rebase.neb.com” onthe world wide web for additional information on the methylationsensitivity of specific restriction endonucleases; see also, Roberts etal., Nucleic Acids Res. 29:268-69, 2001). Thus, while the disclosedmethods and kits have been designed to work with unmodified gDNA, thosein the art will appreciate, that in certain instances the disclosedmethods and kits can be used with such pretreated nucleic acid sequencesalthough pretreatment is not necessary and generally is not useful inimplementing the teaching herein.

In certain embodiments, methods for determining the degree of targetnucleotide methylation are disclosed comprising at least one step forinterrogating at least one target nucleotide; at least one step forgenerating at least one (mis)ligation product; and at least one step fordetermining the degree of methylation of at least one target nucleotide.In certain embodiments, such methods further comprise at least one stepfor generating at least one amplified (mis)ligation product; at leastone step for generating at least one digested (mis)ligation product; orcombinations thereof. Those skilled in the art will appreciate that theat least one step for interrogating can be performed using the probesand probe sets disclosed herein; that the at least one step forgenerating at least one (mis)ligation product can be performed using theligation means and/or ligation techniques disclosed herein; that the atleast one step for generating at least one amplified (mis)ligationproduct can be performed using the amplification means, amplificationtechniques, ligation means, and/or ligation techniques disclosed herein,including combinations thereof; that the at least one step forgenerating at least one digested (mis)ligation product can be performedusing the digesting means and/or digestion techniques disclosed herein;and that the at least one step for determining the degree of methylationof at least one target nucleotide can be performed using the determiningmeans and techniques disclosed herein. In certain embodiments,determining can, but need not, comprise substeps for separating,detecting, and/or analyzing/comparing. In certain embodiments, theseparating is performed independently, i.e., is not a substep of thedetermining. Certain of the disclosed methods and kits comprise at leasttwo separating steps and can, but need not, include at least twoseparating technique.

Kits for determining the degree of methylation of at least one targetnucleotide are also provided. Kits serve to expedite the performance ofthe disclosed methods by assembling two or more components required forcarrying out the methods. Kits generally contain components inpre-measured unit amounts to minimize the need for measurements byend-users. Kits preferably include instructions for performing one ormore of the disclosed methods. Typically, the kit components areoptimized to operate in conjunction with one another.

In certain embodiments, kits comprise at least one probe, at least oneprobe set, at least one primer, at least one hybridization tag, at leastone hybridization tag complement, at least one mobility modifier, atleast one reporter probe, at least one affinity tag, or combinationsthereof. In certain embodiments, kits comprise at least one ligationagent, at least one polymerase, at least one nuclease, at least onerestriction enzyme, at least one chemical digestion means, at least onenucleotide, at least one substrate, at least one of reporter group, orcombinations thereof. In certain embodiments, kits are disclosed thatcomprise at least one means for ligating, at least one means foramplifying, at least one means for separating, at least one means fordigesting, at least one detection means, or combinations thereof.

Certain embodiments of the disclosed methods and kits comprise at leastone ligation agent. In certain embodiments, the ligation agent comprisesat least one ligase, such as DNA ligase or RNA ligase, including,without limitation, the bacteriophage T4 (T4) DNA ligase, T4 RNA ligase,E. coli DNA ligase, or E. coli RNA ligase. In certain embodiments atleast one ligase comprises at least one thermostable ligase. Exemplarythermostable ligases include without limitation, Thermus speciesligases, Pfu ligase, Afu ligase, and the like, including ligases ofbacteriophages that infect thermophilic or hyperthermophilic eubacteriaand viruses that infect archaea, formerly known as archaebacteria. For adescription of Afu ligase, see co-filed U.S. Provisional PatentApplication Ser. No. 60/567,120, filed Apr. 30, 2004, for “Compositions,Methods, and Kits for (Mis)ligating Oligonucleotides, by Karger et al.and co-filed U.S. Patent Provisional Application Ser. No. 60/567,068,filed Apr. 30, 2004 for “Methods and Kits for Identifying TargetNucleotides in Mixed Populations,” by Karger et al.

In certain embodiments, ligation is performed non-enzymatically. Whilenot limiting, non-enzymatic ligation typically includes bothphotoligation and chemical ligation, such as, autoligation and ligationin the presence of an “activating” and/or reducing agent. Non-enzymaticligation can utilize specific reactive groups on the respective 3′ and5′ ends of the probes to be ligated. Thus, in certain embodiments of thedisclosed methods and kits, the ligation agent comprises, one or more“activating” or reducing agent. In certain embodiments, the at least oneligation agent comprises at least one photoligation source. In certainembodiments, one or more probes suitable for ligation are provided thatcomprise appropriate reactive groups for non-enzymatic ligation. Thus,the disclosed ligation means comprise a wide range of enzymatic,chemical and photochemical techniques and reagents for joining the endsof suitable probes.

In certain embodiments the disclosed methods and kits further compriseat least one amplifying means, for example at least one polymerase,including, but not limited to at least one DNA polymerase, at least oneRNA polymerase, at least one reverse transcriptase, or combinationsthereof. Such polymerases provide a means for amplifying at least onenucleotide. Exemplary polymerases include DNA polymerase I, T4 DNApolymerase, SP6 RNA polymerase, T3 RNA polymerase, T7 RNA polymerase,AMV reverse transcriptase, M-MLV reverse transcriptase, and the like. Incertain embodiments, at least one DNA polymerase lacks 5′->3′exonuclease activity, for example, but not limited to Klenow fragment ofDNA polymerase, 9° N_(m)™ DNA polymerase, Vent_(R)® (exo⁻) DNApolymerase, Deep Vent_(R)® (exo) DNA polymerase, Therminator™ DNApolymerase, and the like. In certain embodiments, at least onepolymerase is thermostable. Exemplary thermostable polymerases includeTaq polymerase, Tfl polymerase, Tth polymerase, Tli polymerase, Pfupolymerase, AmpliTaq Gold® polymerase, 9° N_(m)™ DNA polymerase,Vent_(R)® DNA polymerase, Deep Vent_(R)® DNA polymerase, UlTmapolymerase, and the like.

In certain embodiments, the disclosed methods and kits comprise at leastone digestion means, for example but not limited to enzymatic andchemical means for digesting at least part of at least one probe, atleast part of at least one (mis)ligation product, at least part of atleast one amplified (mis)ligation product, or combinations thereof.Exemplary enzymatic means for performing a digestion step includewithout limitation nucleases, for example but not limited to,endonucleases and exonucleases, such as BAL-31 nuclease, mung beannuclease, exonuclease I, exonuclease III, λ exonuclease, T7 exonuclease,exonuclease T, recJ, and RNase H; restriction enzymes; and the like,including enzymatically active variants or mutants thereof. An alkalinehydrolysis step for digesting the RNA portion of at least one RNA-DNAhybrid or RNA:DNA duplex is one example of chemical digestion means.

The skilled artisan will understand that any of a number of nucleases,polymerases, and ligases could be used in the methods and kits of theinvention, including without limitation, those isolated fromthermostable or hyperthermostable prokaryotic, eukaryotic, or archaelorganisms. The skilled artisan will also understand the terms “ligase”,“nuclease” and “polymerase” include not only naturally occurringenzymes, but also recombinant enzymes; and enzymatically activefragments, cleavage products, mutants, or variants of such enzymes, forexample but not limited to Klenow fragment, Stoffel fragment, Taq FS(Applied Biosystems, Foster City, Calif.), 9° N_(m)™ DNA Polymerase (NewEngland BioLabs, Beverly, Mass.), and mutant enzymes described in Luoand Barany, Nucl. Acids Res. 24:3079-3085 (1996), and U.S. Pat. Nos.6,265,193 and 6,576,453. Reversibly modified nucleases, ligases, andpolymerases, for example but not limited to those described in U.S. Pat.No. 5,773,258, are also within the scope of the disclosed teachings.Those in the art will understand that any protein with the desiredenzymatic activity, be it ligating, amplifying, or digesting, can beused in the disclosed methods and kits. Descriptions of nucleases,ligases, and polymerases can be found in, among other places, Twyman,Advanced Molecular Biology, BIOS Scientific Publishers (1999); EnzymeResource Guide, rev. 092298, Promega (1998); Sambrook and Russell,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, 3d ed.(2001)(hereinafter “Sambrook and Russell”); Sambrook, Fritsch, andManiatis, Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, 2d ed. (1989)(hereinafter “Sambrook et al.”); Ausbel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(including supplements through the March 2004)(hereinafter “Ausbel etal.”).

In certain embodiments, the methods and kits disclosed herein compriseat least one polymerase, at least one ligation agent, at least onedigestion agent, or combinations thereof. In certain embodiments, themethods disclosed herein comprise ligation reactions and can furthercomprise primer extension, including but not limited to “gap filling”reactions and the polymerase chain reaction (PCR); transcription,including but not limited to reverse transcription; digestion reactions,including enzymatic or chemical digesting agents; or combinationsthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematically depicts an illustrative competing ligationreaction comprising two probe sets. The target nucleotide (in thisexample the nucleotide “C”) in the target nucleic acid sequence isunderlined (top line). Probe set 1 comprises an upstream probe with a3′-end comprising the nucleotides —C-G and a downstream probe comprisinga 5′-end comprising the nucleotides T-A-C— (middle line). The ligationsite for probe set 1 is between G and T, as shown by arrow 1. Probe set2 comprises an upstream probe with a 3′-end comprising the nucleotides—C-G-T-A and a downstream probe with a 5′-end comprising the nucleotideC— (bottom line). The ligation site for probe set 2 is between the A andthe second C (left to right), as shown by arrow 2.

FIG. 2: Schematically depicts an exemplary misligation reactioncomprising an upstream probe with a 3′-end comprising —C—H and adownstream probe with a 5′-end comprising A-. The ligation site is shownwith an arrow and the target nucleotide is underlined. H represents anyof A, C, T, or U, including but not limited to analogs and Modificationsthereof; but not G.

FIG. 3: Schematically depicts an exemplary misligation reaction withprobes from two competing probe sets. The target nucleotide in thetarget nucleic acid sequence is underlined. The 3′-end of the upstreamprobe for probe set 1 comprises the nucleotide -T and the 5′-end of thedownstream probe comprises B—C-G-T-T-C—. B represents any of C, G, T, orU, including but not limited to analogs and Modifications thereof; butnot A. The 3′-end of the upstream probe for probe set 2 comprises thenucleotides -G-T-V and the 5′-end of the downstream probe comprises C—.V represents any of A, C, or G, including but not limited to analogs andModifications thereof; but not T or U. The ligation sites for probe sets1 and 2 are shown by arrows 1 and 2, respectively.

FIG. 4: Depicts an electropherogram showing ligation product peaksobtained from an illustrative ligation assay, described in Example 1.The upper panel shows the results obtained using a non-methylatedsynthetic model template (“Template”) and the lower panel shows theresults obtained when the synthetic model template comprised5-methylcytosine as the target nucleotide (“^(Me)Template”). The peakcorresponding to the ligation product of Probe Set 1 is marked “1”, thepeak corresponding to the ligation product of Probe Set 2 is marked “2”,and the peak corresponding to the ligation product of Probe Set 3 ismarked “3”. The peak marked 4 is the internal size standard.

FIGS. 5A-C: Depict electropherograms showing misligation product peaksobtained from an exemplary competitive ligation assay, described inExample 2. The upper panels show the results obtained using anon-methylated synthetic P16 template (“Template”) and the lower panelsand the lower panels show the results obtained when the synthetic P16template comprised 5-methylcytosine as the target nucleotide(“^(Me)Template”). The peak corresponding to the misligation productgenerated using ligation probes 8 and 10 is marked “LP 8-10”, the peakcorresponding to the misligation product generated using ligation probes9 and 10 is marked “LP 9-10”, and the peak corresponding to themisligation product generated using ligation probes 10 and 11 is marked“LP 11-10”.

FIGS. 6A-C: depict electropherograms showing misligation product peaksobtained from an exemplary misligation assay, described in Example 3.The peak corresponding to the misligation product generated usingligation probes 13 and 16 is marked “LP 13-16”, the peak correspondingto the misligation product generated using ligation probes 14 and 16 ismarked “LP 14-16”, and the peak corresponding to the misligation productgenerated using ligation probes 15 and 16 is marked “LP 15-16”. Theupper panels show the results obtained using non-methylated templates(“Template”) and the lower panels show the results obtained usingmethylated templates (“^(Me)Template”).

FIGS. 7A-C: Depict electropherograms showing misligation product peaksobtained from an exemplary competitive ligation assay, described inExample 4. The upper panels show the results obtained using anon-methylated synthetic E2F2 template (“Template”) and the lower panelsshow the results obtained when the synthetic E2F2 template comprised5-methylcytosine as the target nucleotide (“^(Me)Template”). The peakcorresponding to the misligation product generated using ligation probes21 and 22 is marked “LP 21-22”, the peak corresponding to themisligation product generated using ligation probes 22 and 23 is marked“LP 22-23”, the peak corresponding to the misligation product peakgenerated using ligation probes 22 and 24 is marked “LP 22-24”.

FIGS. 8A-B: Depict electropherograms showing misligation product peaksobtained from an exemplary competitive misligation assay, described inExample 5. The upper panels show the results obtained using anon-methylated synthetic E2F2 template (“Template”) and the lower panelsshow the results obtained when the synthetic E2F2 template comprised5-methylcytosine as the target nucleotide (“^(Me)Template”). The peakcorresponding to the misligation product generated using ligation probes22* and 21 is marked “LP 22*-21”, the peak corresponding to themisligation product generated using ligation probes 22* and 23 is marked“LP 22*-23”, and the peak corresponding to the misligation productgenerated using ligation probes 22* and 24 is marked “LP 22*-24”.

FIGS. 9A-C: Depict electropherograms showing the peaks obtained from anexemplary competitive misligation assay described in Example 6. Theupper panel shows the misligation product surrogate peak heightsobtained using non-methylated gDNA (“gDNA”) and the lower panel showsthe misligation product surrogate peak heights obtained using methylatedgDNA (“^(Me)gDNA”). The detected peak corresponding to the misligationproduct surrogate generated using ligation probes 25 and 26 is marked“LPS 25-26”, the detected peak corresponding to the misligation productsurrogate generated using probes 25 and 27 is marked “LP 25-27”, and soforth.

FIGS. 10A-C: Depicts electropherograms showing the peaks obtained froman exemplary competitive misligation assay described in Example 7. Theupper panels show the peaks obtained using “gDNA” and the lower panelsshow the peaks obtained using “^(Me)gDNA”. The detected peakcorresponding to the misligation product surrogate generated usingprobes 25 and 31 is marked “LPS 25-31”, the detected peak correspondingto the misligation product surrogate generated using probes 25 and 32 ismarked “LP 25-32”, and so forth.

FIGS. 11A-C: Depict electropherograms showing the peaks obtained from anexemplary competitive misligation assay described in Example 8. Theupper panels show the peaks obtained using gDNA and the lower panelsshow the peaks obtained using methylated gDNA (^(Me)gDNA). The detectedpeak corresponding to the misligation product surrogates generated usingprobes 36 and 37 is marked “LPS 36-37”; the detected peak correspondingto misligation product surrogate generated using probes 38 and 37 ismarked “LP 38-37”, and so forth.

FIGS. 12A-D: depict electropherograms showing the ligation product peaksobtained from an illustrative analysis of four ligases in an exemplarymethylation detection ligation assay, described in Example 10. The upperpanels show the ligation product peaks LP 2-3 (probe set 1), LP 4-5(probe set 2), and LP 6-7 (probe set 3) obtained using non-methylatedtemplate and the lower panels show the results obtained using themethylated synthetic template. FIG. 12A depicts the results obtainedusing Afu ligase; FIG. 12B depicts the results obtained using Thermussp. AK16D ligase; FIG. 12C depicts the results obtained using Tthligase; and FIG. 12D depicts the results obtained using Taq ligase.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the described subject matter inany way. All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and Internet web pages are expressly incorporated byreference in their entirety for any purpose. In the event that one ormore of the incorporated literature and similar materials conflicts withor contradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

I. DEFINITIONS

The term “affinity tag” as used herein refers to at least one componentof a multi-component complex, wherein the components of themulti-component complex specifically interact with or bind to eachother, for example but not limited to a capture moiety and itscorresponding capture ligand. Exemplary multiple-component complexesinclude without limitation, ligands and their receptors, including butnot limited to, avidin-biotin, streptavidin-biotin, and derivatives ofbiotin, streptavidin and/or avidin, including but not limited todesthiobiotin, NeutrAvidin (Molecular Probes, Eugene, Oreg.), CaptAvidin(Molecular Probes), and the like; binding proteins/peptides, includingbut not limited to maltose-maltose binding protein (MBP),calcium-calcium binding protein/peptide (CBP); antigen-antibody,including but not limited to epitope tags, including but not limited toc-MYC (e.g., EQKLISEEDL), HA (e.g., YPYDVPDYA), VSV-G (e.g.,YTDIEMNRLGK), HSV (e.g., QPELAPEDPED), V5 (e.g., GKPIPNPLLGLDST), andFLAG Tag™ (e.g., DYKDDDDKG), and their corresponding anti-epitopeantibodies; haptens, for example but not limited to dinitrophenyl anddigoxigenin, and their corresponding antibodies; aptamers and theircorresponding targets; poly-His tags (e.g., penta-His and hexa-His) andtheir binding partners, including without limitation, correspondingimmobilized metal ion affinity chromatography (IMAC) materials andanti-poly-His antibodies; fluorophores and anti-fluorophore antibodies;and the like. In certain embodiments, affinity tags are used as at leastpart of a means for separating, as at least part of a means fordetecting, or as at least part of: a means for separating and as a meansfor detecting.

The terms “annealing” and “hybridization” are used interchangeably andmean the base-pairing interaction of one nucleic acid with anothernucleic acid that results in formation of a duplex, triplex, or otherhigher-ordered structure. In certain embodiments, the primaryinteraction is base specific, e.g., A:T, A:U and G:C, by Watson/Crickand Hoogsteen-type hydrogen bonding. In certain embodiments,base-stacking and hydrophobic interactions may also contribute to duplexstability. Conditions for hybridizing nucleic acid probes and primers tocomplementary and substantially complementary target sequences are wellknown, e.g., as described in Nucleic Acid Hybridization, A PracticalApproach, B. Hames and S. Higgins, eds., IRL Press, Washington, D.C.(1985) and J. Wetmur and N. Davidson, Mol. Biol. 31:349 et seq. (1968).In general, whether such annealing takes place is influenced by, amongother things, the length of the probes and the complementary targetsequences, the pH, the temperature, the presence of mono- and divalentcations, the proportion of G and C nucleotides in the hybridizingregion, the viscosity of the medium, and the presence of denaturants.Such variables influence the time required for hybridization. Thus, thepreferred annealing conditions will depend upon the particularapplication. Such conditions, however, can be routinely determined bypersons of ordinary skill in the art, without undue experimentation.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, BAC, ACB, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, MA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

The term “corresponding” as used herein refers to at least one specificrelationship between the elements to which the term refers. For example,at least one first probe of a particular probe set corresponds to atleast one second probe of the same probe set, and vice versa. At leastone primer is designed to anneal with the primer-binding portion of atleast one corresponding probe, at least one corresponding (mis)ligationproduct, at least one corresponding amplified (mis)ligation product, atleast one corresponding digested (mis)ligation product, at least onecorresponding digested amplified (mis)ligation product, or combinationsthereof. The target-specific portions of the probes of a particularprobe set are designed to hybridize with a complementary orsubstantially complementary region of the corresponding target nucleicacid sequence. A particular affinity tag binds to the correspondingaffinity tag, for example but not limited to, biotin binding tostreptavidin. A particular hybridization tag anneals with itscorresponding hybridization tag complement; and so forth.

The term “enzymatically active mutants or variants thereof” when used inreference to one or more enzyme, such as one or more polymerase, one ormore ligase, one or more nuclease, or the like, refers to one or morepolypeptide derived from the corresponding enzyme that retains at leastsome of the desired enzymatic activity, such as ligating, amplifying, ordigesting, as appropriate. Also within the scope of this term are:enzymatically active fragments, including but not limited to, cleavageproducts, for example but not limited to Klenow fragment, Stoffelfragment, or recombinantly expressed fragments and/or polypeptides thatare smaller in size than the corresponding enzyme; mutant forms of thecorresponding enzyme, including but not limited to, naturally-occurringmutants, such as those that vary from the “wild-type” or consensus aminoacid sequence, mutants that are generated using physical and/or chemicalmutagens, and genetically engineered mutants, for example but notlimited to random and site-directed mutagenesis techniques; amino acidinsertions and deletions, and changes due to nucleic acid nonsensemutations, missense mutations, and frameshift mutations (see, e.g.,Sriskanda and Shuman, Nucl. Acids Res. 26(2):525-31, 1998; Odell et al.,Nucl. Acids Res. 31(17):5090-5100, 2003); reversibly modified nucleases,ligases, and polymerases, for example but not limited to those describedin U.S. Pat. No. 5,773,258; biologically active polypeptides obtainedfrom gene shuffling techniques (see, e.g., U.S. Pat. Nos. 6,319,714 and6,159,688), splice variants, both naturally occurring and geneticallyengineered, provided that they are derived, at least in part, from oneor more corresponding enzymes; polypeptides corresponding at least inpart to one or more such enzymes that comprise modifications to one ormore amino acids of the native sequence, including without limitation,adding, removing or altering glycosylation, disulfide bonds, hydroxylside chains, and phosphate side chains, or crosslinking, provided suchmodified polypeptides retain at least some of the desired catalyticactivity; and the like.

The skilled artisan will readily be able to measure enzymatic activityusing an appropriate assay known in the art. Thus, an appropriate assayfor polymerase catalytic activity might include, for example, measuringthe ability of a variant to incorporate, under appropriate conditions,rNTPs or dNTPs into a nascent polynucleotide strand in atemplate-dependent manner. Likewise, an appropriate assay for ligasecatalytic activity might include, for example, the ability to ligateadjacently hybridized oligonucleotides comprising appropriate reactivegroups, such as disclosed herein. Protocols for such assays may befound, among other places, in Sambrook et al., Sambrook and Russell,Ausbel et al., and Housby and Southern, Nucl. Acids Res. 26:4259-66,1998).

The terms “fluorophore” and “fluorescent reporter group” are intended toinclude any compound, label, or moiety that absorbs energy, typicallyfrom an illumination source or energy transfer, to reach anelectronically excited state, and then emits energy, typically at acharacteristic wavelength, to achieve a lower energy state. For examplebut without limitation, when certain fluorophores are illuminated by anenergy source with an appropriate excitation wavelength, typically anincandescent or laser light source, photons in the fluorophore areemitted at a characteristic fluorescent emission wavelength.Fluorophores, sometimes referred to as fluorescent dyes, may typicallybe divided into families, such as fluorescein and its derivatives;rhodamine and its derivatives; cyanine and its derivatives; coumarin andits derivatives; Cascade Blue™ and its derivatives; Lucifer Yellow andits derivatives; BODIPY and its derivatives; and the like. Exemplaryfluorophores include indocarbocyanine (C3), indodicarbocyanine (C5),Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red, Pacific Blue, Oregon Green 488,Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568,Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, JOE,Lissamine, Rhodamine Green, BODIPY, fluorescein isothiocyanate (FITC),carboxy-fluorescein (FAM), phycoerythrin, rhodamine, dichlororhodamine(dRhodamine™), carboxy tetramethylrhodamine (TAMRAT™),carboxy-X-rhodamine (ROX™), LIZ™, VIC™, NED™, PET™, SYBR, PicoGreen,RiboGreen, and the like. Descriptions of fluorophores and their use, canbe found in, among other places, R. Haugland, Handbook of FluorescentProbes and Research Products, 9^(th) ed. (2002), Molecular Probes,Eugene, Oreg. (hereinafter “Molecular Probes Handbook”); M. Schena,Microarray Analysis (2003), John Wiley & Sons, Hoboken, N.J.; SyntheticMedicinal Chemistry 2003/2004 Catalog, Berry and Associates, Ann Arbor,Mich.; U.S. Pat. No. 6,025,505; G. Hermanson, Bioconjugate Techniques,Academic Press (1996; hereinafter “Bioconjugate Techniques”); and GlenResearch 2002 Catalog, Sterling, Va. Near-infrared dyes are expresslywithin the scope of the terms fluorophore and fluorescent reportergroup, as are combination labels, such as combinatorial fluorescenceenergy transfer tags (see, e.g. Tong et al., Nat. Biotech. 19:756-59,2001).

The terms “groove binder” and “minor groove binder” refer to smallmolecules that fit into the minor groove of double-stranded DNA,typically in a sequence specific manner. Generally, minor groove bindersare long, flat molecules that can adopt a crescent-like shape and thus,snugly fit into the minor groove of a double helix, often displacingwater. Minor groove binding molecules typically comprise severalaromatic rings connected by bonds with torsional freedom, such as butnot limited to, furan, benzene, or pyrrole rings. Exemplary minor groovebinders include without limitation, antibiotics such as netropsin,distamycin, berenil, pentamidine and other aromatic diamidines, Hoechst33258, SN 6999, aureolic anti-tumor drugs such as chromomycin andmithramycin, CC-1065, dihydrocyclopyrroloindole tripeptide (DPI₃),1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPl₃), and relatedcompounds and analogues. In certain embodiments, at least one probe, atleast one primer, at least one reporter probe, or combinations thereof,comprises at least one minor groove binder. Detailed descriptions ofminor groove binders can be found in, among other places, Nucleic Acidsin Chemistry and Biology, 2d ed., Blackburn and Gait, eds., OxfordUniversity Press, 1996 (hereinafter “Blackburn and Gait”), particularlyin section 8.3; Kumar et al., Nucl. Acids Res. 26:831-38, 1998; Kutyavinet al., Nucl. Acids Res. 28:655-61, 2000; Turner and Denny, Curr. DrugTargets 1:1-14, 2000; Kutyavin et al., Nucl. Acids Res. 25:3718-25,1997; Lukhtanov et al., Bioconjug. Chem. 7:564-7, 1996; Lukhtanov etal., Bioconjug. Chem. 6: 418-26, 1995; U.S. Pat. No. 6,426,408; and PCTPublished Application No. WO 03/078450. Primers and reporter probescomprising minor groove binders are commercially available from, amongother places, Applied Biosystems and Epoch Biosciences, Bothell, Wash.

The term “hybridization tag” as used herein refers to an oligonucleotidesequence that can be used for separating the element (e.g.,(mis)ligation products, (mis)ligation product surrogates, ZipChutes™,etc.) of which it is a component or to which it is bound, includingwithout limitation, bulk separation; for tethering or attaching theelement to which it is bound to a substrate, which may or may notinclude separating; for annealing a hybridization tag complement thatmay comprise at least one moiety, such as a mobility modifier, one ormore reporter groups, and the like; or combinations thereof. In certainembodiments, the same hybridization tag is used with a multiplicity ofdifferent elements to effect: bulk separation, substrate attachment, orcombinations thereof. A “hybridization tag complement” typically refersto at least one oligonucleotide that comprises at least one sequence ofnucleotides that are at least substantially complementary to andhybridize with the corresponding hybridization tag. In variousembodiments, hybridization tag complements serve as capture moieties forattaching at least one hybridization tag:element complex to at least onesubstrate; serve as “pull-out” sequences for bulk separation procedures;or both as capture moieties and as pull-out sequences. In certainembodiments, at least one hybridization tag complement comprises atleast one reporter group and serves as a label for at least one(mis)ligation product, at least one (mis)ligation product surrogate, orcombinations thereof. In certain embodiments, determining comprisesdetecting one or more reporter groups on or attached to at least onehybridization tag complement or at least part of a hybridization tagcomplement.

Typically, hybridization tags and their corresponding hybridization tagcomplements are selected to minimize: internal self-hybridization;cross-hybridization with different hybridization tag species, nucleotidesequences in a reaction composition, including but not limited to gDNA,different species of hybridization tag complements, target-specificportions of probes, and the like; but should be amenable to facilehybridization between the hybridization tag and its correspondinghybridization tag complement. Hybridization tag sequences andhybridization tag complement sequences can be selected by any suitablemethod, for example but not limited to, computer algorithms such asdescribed in PCT Publication Nos. WO 96/12014 and WO 96/41011 and inEuropean Publication No. EP 799,897; and the algorithm and parameters ofSantaLucia (Proc. Natl. Acad. Sci. 95:1460-65 (1998)). Descriptions ofhybridization tags can be found in, among other places, U.S. Pat. Nos.6,309,829 (referred to as “tag segment” therein); 6,451,525 (referred toas “tag segment” therein); 6,309,829 (referred to as “tag segment”therein); 5,981,176 (referred to as “grid oligonucleotides” therein);5,935,793 (referred to as “identifier tags” therein); and PCTPublication No. WO 01/92579 (referred to as “addressablesupport-specific sequences” therein); and Gerry et al., J. Mol. Biol.292:251-262 (1999; referred to as “zip-codes” and “zip-code complements”therein). Those in the art will appreciate that a hybridization tag andits corresponding hybridization tag complement are, by definition,complementary to each other and thus the terms hybridization tag andhybridization tag complement are relative and can typically be usedinterchangeably in most contexts.

Hybridization tags can be located on at least one end of at least oneprobe, at least one primer, at least one (mis)ligation product, at leastone (mis)ligation product surrogate, or combinations thereof; or theycan be located internally. In certain embodiments, at least onehybridization tag is attached to at least one probe, at least oneprimer, at least one (mis)ligation product, at least one (mis)ligationproduct surrogate, or combinations thereof, via at least one linker arm.In certain embodiments, at least one linker arm is cleavable.

In certain embodiments, hybridization tags are at least 12 bases inlength, at least 15 bases in length, 12-60 bases in length, or 15-30bases in length.

In certain embodiments, at least one hybridization tag is 12, 15, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, or 60 bases in length. Incertain embodiments, at least two hybridization tag:hybridization tagcomplement duplexes have melting temperatures that fall within a Δ T_(m)range (T_(max)-T_(min)) of no more than 10° C. of each other. In certainembodiments, at least two hybridization tag:hybridization tag complementduplexes have melting temperatures that fall within a Δ T_(m) range of5° C. or less of each other.

In certain embodiments, at least one hybridization tag complementcomprises at least one reporter group, at least one mobility modifier,at least one reporter probe-binding portion, or combinations thereof. Incertain embodiments, at least one hybridization tag complement isannealed to at least one corresponding hybridization tag and,subsequently, at least part of that hybridization tag complement isreleased and detected.

The term “ligation product” refers to a molecule that is generated whenan internucleotide linkage is formed between two corresponding probes bythe action of one or more ligation agents. Those in the art understandthat, under certain conditions, such an internucleotide linkage can beformed between: (i) at least one pair of matched probes (i.e., thetarget-specific portions of both probes are fully complementary with thecorresponding sequences of the target), or (ii) at least one pair ofmismatched probes (that is at least one of the two probes comprises atleast one nucleotide or nucleotide analog that is mismatched with thecorresponding template or at least one Modification). Thus, the term(mis)ligation is used herein to collectively refer to at least one matchligation, at least one mismatch ligation (sometimes referred to asmisligation), or at least one match ligation and at least onemisligation. Hence, by way of illustration but without limitation, atleast one “(mis)ligation product” refers to at least one ligationproduct, at least one misligation product, or at least one ligationproduct and at least one misligation product; at least one“(mis)ligation product surrogate” refers to at least one ligationproduct surrogate, at least one misligation product surrogate, or atleast one ligation product surrogate and at least one misligationproduct surrogate; and so forth. The term “misligation” is generallyintended to refer to products, surrogates, and the like that result frommismatch ligation reaction, but not match ligation reactions.

The term “ligation product surrogate” as used herein refers to anymolecule or moiety whose detection or identification indicates theexistence of one or more corresponding ligation products. Exemplaryligation product surrogates include but are not limited to, digestedligation products; amplified ligation products; digested amplifiedligation products; one or more moieties cleaved or released from aligation product or ligation product surrogate; one or morecomplementary strand or counterpart of a ligation product or ligationproduct surrogate; reporter probes, including but not limited tocleavage and amplification products thereof; hybridization tagcomplements, including but not limited to ZipChutes™ (typically amolecule or complex comprising at least one hybridization tagcomplement, at least one mobility modifier, and at least one reportergroup, generally a fluorescent reporter group; see, e.g., AppliedBiosystems Part Number 4344467 Rev. C; see also U.S. Provisional PatentApplication Ser. No. 60/517,470); and the like. The term “digestedamplified ligation product” is intended to include a ligation productthat is digested then amplified as well as a ligation product that isamplified then digested.

As used herein, “ligation rate” or “rate” are relative terms that aredetermined by evaluating at least one measurable parameter of at leastone (mis)ligation product or its surrogate. In certain embodiments, a“ligation rate ratio” or “ratio” is obtained by comparing at least onequantifiable parameter of at least one first (mis)ligation product withthe same measurable parameter of at least one second (mis)ligationproduct generated under the same conditions. By way of illustration,without limitation, if the integrated area under the curve correspondingto exemplary (mis)ligation product A is 10 and the integrated area underthe curve corresponding to exemplary (mis)ligation product B generatedunder the same conditions is 1, the corresponding ligation rate ratio is10:1 (NB) or 1:10 (B/A). In certain embodiments, the ligation rate for agiven ligation product is compared to at least one correspondingstandard curve. Those in the art appreciate that numerous measurableparameters exist that can be used to compare the amounts of two or more(mis)ligation products generated under the same conditions, includingwithout limitation, (mis)ligation product peak height, integrated areaunder the curve for the (mis)ligation products, and so forth. Byevaluating the ligation rate or the ligation rate ratio, one candetermine the degree of methylation for at least one target nucleotide.

The term “mobility-dependent analytical technique” as used herein refersto any means for separating different molecular species based ondifferential rates of migration of those different molecular species inone or more separation techniques. Exemplary mobility-dependent analysistechniques include electrophoresis, chromatography, mass spectroscopy,sedimentation, e.g., gradient centrifugation, field-flow fractionation,multi-stage extraction techniques and the like. Descriptions ofmobility-dependent analytical techniques can be found in, among otherplaces, U.S. Pat. Nos. 5,470,705, 5,514,543, 5,580,732, 5,624,800, and5,807,682; PCT Publication No. WO 01/92579; D. R. Baker, CapillaryElectrophoresis, Wiley-Interscience (1995); Biochromatography: Theoryand Practice, M. A. Vijayalakshmi, ed., Taylor & Francis, London, U.K.(2003); Krylov and Dovichi, Anal. Chem. 72:111 R-128R (2000); Swinneyand Bornhop, Electrophoresis 21:1239-50 (2000); Crabtree et al.,Electrophoresis 21:1329-35 (2000); and A. Pingoud et al., BiochemicalMethods: A Concise Guide for Students and Researchers, Wiley-VCH VerlagGmbH, Weinheim, Germany (2002).

The term “mobility modifier” as used herein refers to at least onemolecular entity, for example but not limited to, at least one polymerchain, that when added to at least one element (e.g., at least oneprobe, at least one primer, at least one (mis)ligation product, at leastone (mis)ligation product surrogate, or combinations thereof) affectsthe mobility of the element to which it is hybridized or bound,covalently or non-covalently, in at least one mobility-dependentanalytical technique. Typically, a mobility modifier changes thecharge/translational frictional drag when hybridized or bound to theelement; or imparts a distinctive mobility, for example but not limitedto, a distinctive elution characteristic in a chromatographic separationmedium or a distinctive electrophoretic mobility in a sieving matrix ornon-sieving matrix, when hybridized or bound to the correspondingelement; or both (see, e.g., U.S. Pat. Nos. 5,470,705 and 5,514,543;Grossman et al., Nucl. Acids Res. 22:4527-34 (1994)). In certainembodiments, a multiplicity of probes exclusive of mobility modifiers, amultiplicity of primers exclusive of mobility modifiers, a multiplicityof (mis)ligation products exclusive of mobility modifiers, amultiplicity of (mis)ligation product surrogates exclusive of mobilitymodifiers, or combinations thereof, have the same or substantially thesame mobility in at least one mobility-dependent analytical technique.

In certain embodiments, a multiplicity of probes, a multiplicity ofprimers, a multiplicity of ligation products, a multiplicity of ligationproduct surrogates, or combinations thereof, have substantially similardistinctive mobilities, for example but not limited to, when amultiplicity of elements comprising mobility modifiers havesubstantially similar distinctive mobilities so they can be bulkseparated or they can be separated from other elements comprisingmobility modifiers with different distinctive mobilities. In certainembodiments, a multiplicity of probes comprising mobility modifiers, amultiplicity of primers comprising mobility modifiers, a multiplicity of(mis)ligation products comprising mobility modifiers, a multiplicity of(mis)ligation product surrogates comprising mobility modifiers, orcombinations thereof, have different distinctive mobilities.

In certain embodiments, at least one mobility modifier comprises atleast one nucleotide polymer chain, including without limitation, atleast one oligonucleotide polymer chain, at least one polynucleotidepolymer chain, or both at least one oligonucleotide polymer chain and atleast one polynucleotide polymer chain. For example but not limited to aseries of additional non-target sequence-specific nucleotides in one ormore probes such as “TTTT”, shown in Table 7; or nucleotide spacers (seee.g., Tong et al., Nat. Biotech. 19:756-759 (2001)). In certainembodiments, at least one mobility modifier comprises at least onenon-nucleotide polymer chain. Exemplary non-nucleotide polymer chainsinclude, without limitation, peptides, polypeptides, polyethylene oxide(PEO), or the like. In certain embodiments, at least one polymer chaincomprises at least one substantially uncharged, water-soluble chain,such as a chain composed of one or more PEO units; a polypeptide chain;or combinations thereof.

The polymer chain can comprise a homopolymer, a random copolymer, ablock copolymer, or combinations thereof. Furthermore, the polymer chaincan have a linear architecture, a comb architecture, a branchedarchitecture, a dendritic architecture (e.g., polymers containingpolyamidoamine branched polymers, Polysciences, Inc. Warrington, Pa.),or combinations thereof. In certain embodiments, at least one polymerchain is hydrophilic, or at least sufficiently hydrophilic whenhybridized or bound to an element to ensure that the element-mobilitymodifier is readily soluble in aqueous medium. Where themobility-dependent analytical technique is electrophoresis, in certainembodiments, the polymer chains are uncharged or have a charge/subunitdensity that is substantially less than that of its correspondingelement.

The synthesis of polymer chains useful as mobility modifiers willdepend, at least in part, on the nature of the polymer. Methods forpreparing suitable polymers generally follow well-known polymer subunitsynthesis methods. These methods, which involve coupling ofdefined-size, multi-subunit polymer units to one another, eitherdirectly or through charged or uncharged linking groups, are generallyapplicable to a wide variety of polymers, such as PEO, polyglycolicacid, polylactic acid, polyurethane polymers, polypeptides,oligosaccharides, and nucleotide polymers. Such methods of polymer unitcoupling are also suitable for synthesizing selected-length copolymers,e.g., copolymers of PEO units alternating with polypropylene units.Polypeptides of selected lengths and amino acid composition, eitherhomopolymer or mixed polymer, can be synthesized by standard solid-phasemethods (see, e.g., Int. J. Peptide Protein Res., 35: 161-214 (1990)).

One method for preparing PEO polymer chains having a selected number ofhexaethylene oxide (HEO) units, an HEO unit is protected at one end withdimethoxytrityl (DMT), and activated at its other end with methanesulfonate. The activated HEO is then reacted with a second DMT-protectedHEO group to form a DMT-protected HEO dimer. This unit-addition is thencarried out successively until a desired PEO chain length is achieved(see, e.g., U.S. Pat. No. 4,914,210; see also, U.S. Pat. No. 5,777,096).

As used herein, the term “Modification” refers to at least onesubstituted hydrocarbon, at least one ribonucleotide, at least one amidebond (including but not limited to at least one PNA, at least one pcPNA,or both), at least one nucleotide analog, at least one groove binder, orcombinations thereof. In certain embodiments, at least one probecomprises at least one Modification, sometimes referred to as a“Modified probe.” In certain embodiments, at least one Modificationcomprises at least one structure shown below,

wherein: (a) R₁ comprises at least one hydrogen, alkyl, substitutedalkyl, alkene, substituted alkene, alkyne, substituted alkyne, aromaticring, substituted aromatic ring, heteroaromatic ring, substitutedheteroaromatic ring, halogen, nitro, cyano, oxygen, substituted oxygen,nitrogen, substituted nitrogen, divalent sulfur, substituted divalentsulfur, sulfonate, sulfonate ester, aldehyde, ketone carbon with R₂,carboxylate carbon as carboxylic acid and ester with R₂, or combinationsthereof; (b) R₂, a substituent on R₁, comprises at least one hydrogen,alkyl, substituted alkyl, alkene, substituted alkene, alkyne,substituted alkyne, aromatic ring, substituted aromatic ring,heteroaromatic ring, substituted heteroaromatic ring, halogen, nitro,cyano, alcohol, ether substituted with R₃, amine, secondary, tertiary,and quaternary amines substituted with R₃, amido substituted with R₃,thiol, thioether substituted with R₃, sulfonate, sulfonate estersubstituted with R₃, phosphate and phosphate esters substituted with R₃,phosphonate and phosphonate esters substituted with R₃, aldehyde, ketonesubstituted with R₃, carboxylate, carboxylate esters substituted withR₃, carboxyamides substituted with R₃., or combinations thereof; and (c)R₃, a substituent on R₂, comprises at least one hydrogen, alkyl,substituted alkyl, alkene, substituted alkene, alkyne, substitutedalkyne, aromatic ring, substituted aromatic ring, heteroaromatic ring,substituted heteroaromatic ring, halogen, nitro, cyano, alcohol, etheras defined in R₂, amine, secondary, tertiary, and quaternary amines asdefined in R₂, amido as defined in R₂, thiol, thioether as defined inR₂, sulfonate, sulfonate ester as defined in R₂, phosphate and phosphateesters as defined in R₂, phosphonate and phosphonate esters as definedin R₂, aldehyde, ketone as defined in R₂, carboxylate, carboxylateesters as defined in R₂, carboxyamides as defined in R₂.

The term “nucleotide base”, as used herein, refers to a substituted orunsubstituted aromatic ring or rings. In certain embodiments, thearomatic ring or rings contain at least one nitrogen atom. In certainembodiments, the nucleotide base is capable of forming Watson-Crickand/or Hoogsteen-type hydrogen bonds with a complementary nucleotidebase. Exemplary nucleotide bases and analogs thereof include, but arenot limited to, naturally occurring nucleotide bases adenine, guanine,cytosine, 5 methyl-cytosine, uracil, thymine, and analogs of thenaturally occurring nucleotide bases, including without limitation,7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine,7-deaza-8-azaadenine, N6-Δ2-isopentenyladenine (6iA),N6-Δ2-isopentenyl-2-methylthioadenine (2 ms6iΔ), N2-dimethylguanine(dmG), 7-methylguanine (7mG), inosine, nebularine, 2-aminopurine,2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudouridine,pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine,isoguanine, 7-deazaguanine, 2-thiopyrimidine, 6-thioguanine,4-thiothymine, 4-thiouracil, O6-methylguanine, N6-methyladenine,O4-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil,pyrazolo[3,4-D]pyrimidines (see, e.g., U.S. Pat. Nos. 6,143,877 and6,127,121 and PCT published application WO 01/38584), ethenoadenine,indoles such as nitroindole and 4-methylindole, and pyrroles such asnitropyrrole. Certain exemplary nucleotide bases can be found, e.g., inFasman, 1989, Practical Handbook of Biochemistry and Molecular Biology,pp. 385-394, CRC Press, Boca Raton, Fla., and the references citedtherein.

The term “nucleotide”, as used herein, refers to a compound comprising anucleotide base linked to the C-1′ carbon of a sugar, such as ribose,arabinose, xylose, and pyranose, and sugar analogs thereof. The termnucleotide also encompasses nucleotide analogs. The sugar may besubstituted or unsubstituted. Substituted ribose sugars include, but arenot limited to, those riboses in which one or more of the carbon atoms,for example the 2′-carbon atom, is substituted with one or more of thesame or different, —R, —OR, —NR2 azide, cyanide or halogen groups, whereeach R is independently H, C1-C6 alkyl, C2-C7 acyl, or C5-C14 aryl.Exemplary riboses include, but are not limited to,2′-(C1-C6)alkoxyribose, 2′-(C5-C14)aryloxyribose, 2′,3′-didehydroribose,2′-deoxy-3′-haloribose, 2′-deoxy-3′-fluororibose,2′-deoxy-3′-chlororibose, 2′-deoxy-3′-aminoribose,2′-deoxy-3′-(C1-C6)alkylribose, 2′-deoxy-3′-(C1-C6)alkoxyribose and2′-deoxy-3′-(C5-C14)aryloxyribose, ribose, 2′-deoxyribose,2′,3′-dideoxyribose, 2′-haloribose, 2′-fluororibose, 2′-chlororibose,and 2′-alkylribose, e.g., 2′-O-methyl, 4′-α-anomeric nucleotides,1′-α-anomeric nucleotides, 2′-4′- and 3′-4′-linked and other “locked” or“LNA”, bicyclic sugar modifications (see, e.g., PCT publishedapplication nos. WO 98/22489, WO 98/39352; and WO 99/14226). ExemplaryLNA sugar analogs within a polynucleotide include, but are not limitedto, the structures:

where B is any nucleotide base.

[m]odifications at the 2′- or 3′-position of ribose include, but are notlimited to, hydrogen, hydroxy, methoxy, ethoxy, allyloxy, isopropoxy,butoxy, isobutoxy, methoxyethyl, alkoxy, phenoxy, azido, cyano, amido,imido, amino, alkylamino, fluoro, chloro and bromo. Nucleotides include,but are not limited to, the natural D optical isomer, as well as the Loptical isomer forms (see, e.g., Garbesi Nucl. Acids Res. 21:4159-65(1993); Fujimori (1990) J. Amer. Chem. Soc. 112:7435; Urata, (1993)Nucleic Acids Symposium Ser. No. 29:69-70). When the nucleotide base ispurine, e.g. A or G, the ribose sugar is attached to the N⁹-position ofthe nucleotide base. When the nucleotide base is pyrimidine, e.g. C, T,or U, the pentose sugar is attached to the N¹-position of the nucleotidebase, except for pseudouridines, in which the pentose sugar is attachedto the C5 position of the uracil nucleotide base (see, e.g., Kornbergand Baker, (1992) DNA Replication, 2^(nd) Ed., Freeman, San Francisco,Calif.).

One or more of the pentose carbons of a nucleotide may be substitutedwith a phosphate ester having the formula:

where α is an integer from 0 to 4. In certain embodiments, a is 2 andthe phosphate ester is attached to the 3′- or 5′-carbon of the pentose.In certain embodiments, the nucleotides are those in which thenucleotide base is a purine, a 7-deazapurine, a pyrimidine, or an analogthereof. “Nucleotide 5′-triphosphate” refers to a nucleotide with atriphosphate ester group at the 5′ position, and is sometimes denoted as“NTP”, or “dNTP” and “ddNTP” to particularly point out the structuralfeatures of the ribose sugar. The triphosphate ester group may includesulfur substitutions for the various oxygens, e.g. α-thio-nucleotide5′-triphosphates. Reviews of nucleotide chemistry can be found in, amongother places, Shabarova, Z. and Bogdanov, A. Advanced Organic Chemistryof Nucleic Acids, VCH, New York, 1994; and Blackburn and Gait.

The term “nucleotide analog”, as used herein, refers to embodiments inwhich the pentose sugar and/or the nucleotide base and/or one or more ofthe phosphate esters of a nucleotide may be replaced with its respectiveanalog. In certain embodiments, exemplary pentose sugar analogs arethose described above. In certain embodiments, the nucleotide analogshave a nucleotide base analog as described above. In certainembodiments, exemplary phosphate ester analogs include, but are notlimited to, alkylphosphonates, methylphosphonates, phosphoramidates,phosphotriesters, phosphorothioates, phosphorodithioates,phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates,phosphoroanilidates, phosphoroamidates, boronophosphates, etc., and mayinclude associated counterions.

Also included within the definition of “nucleotide analog” arenucleotide analog monomers that can be polymerized into polynucleotideanalogs in which the DNA/RNA phosphate ester and/or sugar phosphateester backbone is replaced with a different type of internucleotidelinkage. Exemplary polynucleotide analogs include, but are not limitedto, peptide nucleic acids, in which the sugar phosphate backbone of thepolynucleotide is replaced by a peptide backbone comprising at least oneamide bond. (See, e.g., Datar and Kim, Concepts in Applied MolecularBiology, Eaton Publishing, Westborough, Mass., 2003, particularly atpages 74-75; Verma and Eckstein, Ann. Rev. Biochem. 67:99-134, 1998;Goodchild, Bioconj. Chem., 1:165-187, 1990).

As used herein, the terms “polynucleotide”, “oligonucleotide”, “nucleicacid”, and “nucleic acid sequence” are generally used interchangeablyand include single-stranded and double-stranded polymers of nucleotidemonomers, including 2′-deoxyribonucleotides (DNA) and ribonucleotides(RNA) linked by internucleotide phosphodiester bond linkages, orinternucleotide analogs, and associated counter ions, e.g., H+, NH4+,trialkylammonium, tetraalkylammonium, Mg2+, Na+ and the like. A nucleicacid may be composed entirely of deoxyribonucleotides, entirely ofribonucleotides, or chimeric mixtures thereof. The nucleotide monomerunits may comprise any of the nucleotides described herein, including,but not limited to, naturally occurring nucleotides and nucleotideanalogs. Nucleic acids typically range in size from a few monomericunits, e.g. 5-40 when they are sometimes referred to in the art asoligonucleotides, to several thousands of monomeric nucleotide units.Nucleic acid sequence are shown in the 5′ to 3′ orientation from left toright, unless otherwise apparent from the context or expressly indicateddifferently; and in such sequences, “A” denotes deoxyadenosine, “C”denotes deoxycytidine, “G” denotes deoxyguanosine, “T” denotesthymidine, and “U” denotes uridine.

Nucleic acids include, but are not limited to, genomic DNA, cDNA, hnRNA,mRNA, rRNA, tRNA, fragmented nucleic acid, nucleic acid obtained fromsubcellular organelles such as mitochondria or chloroplasts, and nucleicacid obtained from microorganisms or DNA or RNA viruses that may bepresent on or in a biological sample.

Nucleic acids may be composed of a single type of sugar moiety, e.g., asin the case of RNA and DNA, or mixtures of different sugar moieties,e.g., as in the case of RNA/DNA chimeras. In certain embodiments,nucleic acids are ribopolynucleotides and 2′-deoxyribopolynucleotidesaccording to the structural formulae below:

wherein each B is independently the base moiety of a nucleotide, e.g., apurine, a 7-deazapurine, a purine or purine analog substituted with oneor more substituted hydrocarbons, a pyrimidine, a pyrimidine orpyrimidine analog substituted with one or more substituted hydrocarbons,or an analog nucleotide; each m defines the length of the respectivenucleic acid and can range from zero to thousands, tens of thousands, oreven more; each R is independently selected from the group comprisinghydrogen, halogen, —R″, —OR″, and —NR″R″, where each R″ is independently(C1-C6) alkyl, (C2-C7) acyl or (C5-C14) aryl, cyanide, azide, or twoadjacent Rs are taken together to form a bond such that the ribose sugaris 2′,3′-didehydroribose; and each R′ is independently hydroxyl or

where α is zero, one or two.

In certain embodiments of the ribopolynucleotides and2′-deoxyribopolynucleotides illustrated above, the nucleotide bases Bare covalently attached to the C1′ carbon of the sugar moiety aspreviously described.

The terms “nucleic acid”, “nucleic acid sequence”, “polynucleotide”, and“oligonucleotide” can also include nucleic acid analogs, polynucleotideanalogs, and oligonucleotide analogs. The terms “nucleic acid analog”,“polynucleotide analog” and “oligonucleotide analog” are usedinterchangeably and, as used herein, refer to a nucleic acid thatcontains at least one nucleotide analog and/or at least one phosphateester analog and/or at least one pentose sugar analog. Also includedwithin the definition of nucleic acid analogs are nucleic acids in whichthe phosphate ester and/or sugar phosphate ester linkages are replacedwith other types of linkages, such as N-(2-aminoethyl)-glycine amidesand other amides (see, e.g., Nielsen et al., 1991, Science 254:1497-1500; PCT Publication No. WO 92/20702; U.S. Pat. Nos. 5,719,262 and5,698,685); morpholinos (see, e.g., U.S. Pat. No. 5,698,685; U.S. Pat.No. 5,378,841; U.S. Pat. No. 5,185,144); carbamates (see, e.g., Stirchak& Summerton, J. Org. Chem. 52: 4202, 1987); methylene(methylimino) (see,e.g., Vasseur et al, J. Am. Chem. Soc. 114:4006, 1992);3′-thioformacetals (see, e.g., Jones et al., 1993, J. Org. Chem. 58:2983); sulfamates (see, e.g., U.S. Pat. No. 5,470,967);2-aminoethylglycine, commonly referred to as PNA (see, e.g., PCTPublication No. WO 92/20702; Nielsen, Science 254:1497-1500, 1991); andothers (see, e.g., U.S. Pat. No. 5,817,781; Frier & Altman, Nucl. AcidsRes. 25:4429, 1997 and the references cited therein). Phosphate esteranalogs include, but are not limited to, (i) C₁-C₄ alkylphosphonate,e.g. methylphosphonate; (ii) phosphoramidate; (iii) C₁-C₆alkyl-phosphotriester; (iv) phosphorothioate; and (v)phosphorodithioate. See also, Scheit, Nucleotide Analogs, John Wiley,New York, (1980); Englisch, Agnew. Chem. Int. Ed. Engl. 30:613-29, 1991;Agarwal, Protocols for Polynucleotides and Analogs, Humana Press, 1994;and S. Verma and F. Eckstein, Ann. Rev. Biochem. 67:99-134, 1999.

The term “polymerase” is used in a broad sense herein and includesamplifying means such as DNA polymerases, enzymes that typicallysynthesize DNA by incorporating deoxyribonucleotide triphosphates oranalogs in the 5′=>3′ direction in a template-dependent andprimer-dependent manner; RNA polymerases, enzymes that typicallysynthesize RNA by incorporating ribonucleotide triphosphates or analogs,generally in a template-dependent manner; and reverse transcriptases,also known as RNA-dependent DNA polymerases, that synthesize DNA byincorporating deoxyribonucleotide triphosphates or analogs in the 5′=>3′direction in primer-dependent manner, typically using an RNA template.Descriptions of polymerases can be found in, among other places, R. M.Twyman, Advanced Molecular Biology, Bios Scientific Publishers Ltd.(1999); Polymerase Enzyme Resource Guide, Promega, Madison, Wis. (1998);P. C. Turner et al., Instant Notes in Molecular Biology, Bios ScientificPublishers Ltd. (1997); and B. D. Hames et al., Instant Notes inBiochemistry, Bios Scientific Publishers Ltd. (1997).

The term “primer” as used herein refers to an oligonucleotide comprisingat least one region that is complementary or substantially complementaryto the primer-binding portion of at least one probe, at least one(mis)ligation product, at least one (mis)ligation product surrogate, orcombinations thereof, including sequences that are complementary to anyof these, and that can anneal with such primer-binding portions or theircomplements under appropriate conditions. Primers typically serve asinitiation sites for certain amplification techniques, including but notlimited to, primer extension and PCR. A primer that hybridizes with amultiplicity of different probe species, (mis)ligation product species,(mis)ligation product surrogate species, or combinations thereof, isreferred to as a “universal primer”. In certain embodiments, at leastone primer comprises at least one additional component, including butnot limited to, at least one primer-binding portion, at least onereporter probe-binding portion, at least one reporter group, at leastone hybridization tag, at least one mobility modifier, at least oneaffinity tag, or combinations thereof.

The term “probe” as used herein, refers to an oligonucleotide comprisinga target-specific portion that is capable, under appropriate conditions,of hybridizing with at least a part of at least one corresponding targetnucleic acid sequence. As used herein, the terms probe and probesgenerally refer to ligation probes and misligation probes, includingcompeting ligation probes and competing misligation probes, unlessotherwise apparent from the context. A probe may include Watson-Crickbases or modified bases, including but not limited to, the AEGIS bases(from Eragen Biosciences), described, e.g., in U.S. Pat. Nos. 5,432,272;5,965,364; and 6,001,983. Additionally, bases may be joined by a naturalphosphodiester bond or a different chemical linkage. Different chemicallinkages include, but are not limited to, at least one amide linkage orat least one Locked Nucleic Acid (LNA) linkage, described in, e.g.,published PCT Applications WO 00/56748 and WO 00/66604.

Probes typically are part of at least one ligation probe set or at leastone competing ligation probe set, comprising at least one first probeand at least one second probe. In certain embodiments, at least oneprobe comprises at least one nucleotide in its target-specific portionthat is mismatched relative to at least one portion of its correspondingtarget nucleic acid sequence, at least one Modification, or both atleast one mismatched nucleotide and at least one Modification. Incertain embodiments, at least one mismatched nucleotide also comprisesat least one Modification.

In certain embodiments, at least one probe comprises at least oneadditional component, including but not limited to, at least oneprimer-binding portion, at least one reporter probe-binding portion, atleast one reporter group, at least one hybridization tag, at least onemobility modifier, at least one affinity tag, or combinations thereof.In certain embodiments, such additional components are within thetarget-specific portion, coextensive with the target-specific portion,overlaps at least part of the target-specific portion, or combinationsthereof.

The target-specific portions of ligation probes are of sufficient lengthto permit specific annealing to complementary sequences in correspondingtarget nucleic acid sequences. Likewise, primers are of sufficientlength to permit specific annealing to complementary sequences incorresponding (mis)ligation products, corresponding (mis)ligationproduct surrogates, or combinations thereof. The criteria for designingsequence-specific nucleic acid probes (including but not limited toligation probes and reporter probes) and primers are well known to thosein the art. In certain embodiments, at least one probe, at least oneprimer, or at least one probe and at least one primer comprises at leastone region that is fully complementary with the corresponding sequencesin at least one target nucleic acid sequence, at least one (mis)ligationproduct, at least one (mis)ligation product surrogate, or combinationsthereof. In certain embodiments, at least one probe contains at leastone mismatched nucleotide relative to at least one correspondingnucleotide in the target nucleic acid sequence, at least oneModification, at least one additional component, or combinationsthereof. Detailed descriptions of nucleic acid probe and primer designcan be found in, among other places, Diffenbach and Dveksler, PCRPrimer, A Laboratory Manual, Cold Spring Harbor Press (1995); R. Rapley,The Nucleic Acid Protocols Handbook (2000), Humana Press, Totowa, N.J.(hereinafter “Rapley”); Schena; and Kwok et al., Nucl. Acid Res.18:999-1005 (1990). Primer and probe design software programs are alsocommercially available, including without limitation, Primer Express,Applied Biosystems, Foster City, Calif.; Primer Premier and BeaconDesigner software, PREMIER Biosoft International, Palo Alto, Calif.;Primer Designer 4, Sci-Ed Software, Durham, N.C.; Primer Detective,ClonTech, Palo Alto, Calif.; Lasergene, DNASTAR, Inc., Madison, Wis.;Oligo software, National Biosciences, Inc., Plymouth, Minn.; iOligo,Caesar Software, Portsmouth, N.H.; and RTPrimerDB on the world wide webat realtimeprimerdatabase.ht.st or atmedgen31.urgent.be/primerdatabase/index (see also, Pattyn et al., Nucl.Acid Res. 31:122-23, 2003).

A “probe set” according to the present teachings comprises at least onefirst probe and at least one second probe that typically adjacentlyhybridize to the same target sequence, but not always, and are generallyused for interrogating at least one target nucleotide. The first probeof each probe set is designed to hybridize with the downstream region ofthe target sequence in a sequence-specific manner. The second probe inthe probe set is designed to hybridize with the upstream region of thetarget sequence in a sequence-specific manner. The use of the termsfirst and second with respect to probed and primers is to distinguishone from the other and is generally not intended to be limiting. Thesequence-specific portions of these probes are of sufficient length topermit specific annealing with complementary sequences in targets andprimers, as appropriate. In certain embodiments, both the at least onefirst probe and the at least one second probe in a probe set furthercomprise primer-specific portions suitable for hybridizing with primers.

Under appropriate conditions, adjacently hybridized probes can beligated together by one or more ligation agents to form a ligationproduct, provided that they comprise appropriate reactive groups, forexample, without limitation, a free 3′-hydroxyl or 5′-phosphate group.Some probe sets may comprise more than one first probe or more than onesecond probe or both, to aid in determining the degree of methylation atone or more target nucleotide. Certain of the disclosed methods comprisea multiplicity of different probe sets for determining a multiplicity ofdifferent target nucleotides in a multiplex ligation reaction. Certainembodiments comprise at least one multiplex amplification reaction, atleast one multiplex ligation reaction, or at least one multiplexamplification reaction and at least one multiplex ligation reaction. Incertain embodiments, at least one multiplex amplification reaction andat least one multiplex ligation reaction are performed in the same tube.

Those in the art understand that probes and probe sets that are suitablefor use with the disclosed methods and kits can be identifiedempirically using the current teachings and routine methods known in theart, without undue experimentation. For example, suitable probes andprobe sets can be obtained by selecting appropriate target nucleotidesand target nucleotide sequences by searching relevant scientificliterature, including but not limited to appropriate databases (see,e.g., DNA Methylation Database (MethDB), on the web at methdb.de ormethdb.net; CpG Island Searcher, on the web at cpgislands.corn; the NCBIEntrez Nucleotide database), or by experimental analysis. When targetnucleic acid sequences of interest are identified, test probes can besynthesized (and Modified if desired) using well known oligonucleotidesynthesis and organic chemistry techniques (see, e.g., Current Protocolsin Nucleic Acid Chemistry, Beaucage et al., eds., John Wiley & Sons, NewYork, N.Y., including updates through April 2004 (hereinafter “Beaucageet al.”); Blackburn and Gait; Glen Research 2002 Catalog, Sterling, Va.;and Synthetic Medicinal Chemistry 2003/2004, Berry and Associates,Dexter, Mich.). Test probes and/or probe sets are employed in thedisclosed assays using appropriate target sequences and theirsuitablility for interrogating the target nucleotide is evaluated.Standard curves for determining the degree of target nucleotidemethylation can then be generated, if desired, using pre-determinedmixtures of methylated and non-methylated synthetic templates or gDNA asthe target nucleic acid sequences in one or more of the disclosedligation assays under standard conditions. Those in the art are familiarwith generating and using standard curves (see, e.g., Overholtzer etal., Proc. Natl. Sci. 100:11547-52, 2003).

According to certain embodiments, the primer sets comprise at least onefirst primer and at least one second primer. The first primer of aprimer set is designed to hybridize with the complement of the 5′primer-specific portion of a (mis)ligation product, appropriate(mis)ligation product surrogates, or combinations thereof, in asequence-specific manner. The second primer in that primer set isdesigned to hybridize with a 3′ primer-specific portion of the same(mis)ligation product, appropriate (mis)ligation product surrogates, orcombinations thereof, in a sequence-specific manner. In certainembodiments, at least one primer of the primer set further comprises atleast one reporter group, at least one hybridization tag, at least oneaffinity tag, or combinations thereof. Suitable probes and primers canbe synthesized using methods well known on the art. Detaileddescriptions of probe and primer synthesis and phosphorylation can befound in, among other places, Beaucage et al., Tong et al., Nucl. AcidsRes. 27:788-94 (1999), Housby and Southern, Nucl. Acids Res. 26:4259-66(1998), and Grossman et al., Nucl. Acids Res. 22:4527-34 (1994).

The term “reporter group” is used in a broad sense herein and refers toany identifiable tag, label, or moiety. The skilled artisan willappreciate that many different species of reporter groups can be used inthe present teachings, either individually or in combination with one ormore different reporter group. Exemplary reporter groups include, butare not limited to, fluorophores, radioisotopes, chromogens, enzymes,antigens including but not limited to epitope tags, heavy metals, dyes,phosphorescence groups, chemiluminescent groups, electrochemicaldetection moieties, affinity tags, binding proteins, phosphors, rareearth chelates, near-infrared dyes, including but not limited to,“Cy.7.5Ph.NCS,” “Cy.7.OphEt.NCS,” “Cy7.OphEt.CO₂Su”, and IRD800 (see,e.g., J. Flanagan et al., Bioconjug. Chem. 8:751-56 (1997); and DNASynthesis with IRD800 Phosphoramidite, LI-COR Bulletin #111, LI-COR,Inc., Lincoln, Nebr.), electrochemiluminescence labels, including butnot limited to, tris(bipyridal) ruthenium (II), also known as Ru(bpy)₃²⁺, Os(1,10-phenanthroline)₂bis(diphenylphosphino)ethane²⁺, also knownas Os(phen)₂(dppene)²⁺, luminol/hydrogen peroxide,Al(hydroxyquinoline-5-sulfonic acid),9,10-diphenylanthracene-2-sulfonate, andtris(4-vinyl-4′-methyl-2,2′-bipyridal) ruthenium (II), also known asRu(v-bpy₃ ²⁺), and the like.

The term reporter group also encompasses at least one element ofmulti-element indirect reporter systems, including without limitation,affinity tags such as biotin:avidin, antibody:antigen, ligand:reqeptorincluding but not limited to binding proteins and their ligands,enzyme:substrate, and the like, in which one element interacts with oneor more other elements of the system in order to effect the potentialfor a detectable signal. Exemplary multi-element reporter systemsinclude an oligonucleotide comprising at least one biotin reporter groupand a streptavidin-conjugated fluorophore, or vice versa; anoligonucleotide comprising at least one dinitrophenyl (DNP) reportergroup and a fluorophore-labeled anti-DNP antibody; and the like. Incertain embodiments, reporter groups, particularly multi-elementreporter groups, are not necessarily used for detection, but ratherserve as affinity tags for isolation/separation, for example but notlimited to, a biotin reporter group and a streptavidin coated substrate,or vice versa; a digoxygenin reporter group and an anti-digoxygeninantibody or a digoxygenin-binding aptamer; a DNP reporter group and ananti-DNP antibody or a DNP-binding aptamer; and the like. Detailedprotocols for attaching reporter groups to oligonucleotides,polynucleotides, peptides, antibodies and other proteins, mono-, di- andoligosaccharides, organic molecules, and the like can be found in, amongother places, Bioconjugate Techniques; Beaucage et al.; Molecular ProbesHandbook; and Pierce Applications Handbook and Catalog 2003-2004, PierceBiotechnology, Rockford, Ill., 2003 (hereinafter “Pierce ApplicationsHandbook”).

In certain embodiments, at least one reporter group comprises at leastone electrochemiluminescent moiety that can, under appropriateconditions, emit detectable electrogenerated chemiluminescence (ECL). InECL, excitation of the electrochemiluminescent moiety iselectrochemically driven and the chemiluminescent emission can beoptically detected. Exemplary electrochemiluminescent reporter groupspecies include: Ru(bpy)₃ ²⁺ and Ru(v-bpy)₃ ²⁺ with emission wavelengthsof 620 nm; Os(phen)₂(dppene)²⁺ with an emission wavelength of 584 nm;luminol/hydrogen peroxide with an emission wavelength of 425 nm;Al(hydroxyquinoline-5-sulfonic acid) with an emission wavelength of 499nm; and 9,10-diphenylanothracene-2-sulfonate with an emission wavelengthof 428 nm; and the like. Forms of these three electrochemiluminescentreporter group species that are modified to be amenable to incorporationinto probes are commercially available or can be synthesized withoutundue experimentation using techniques known in the art. For example, aRu(bpy)₃ ²⁺N-hydroxy succinimide ester for coupling to nucleic acidsequences through an amino linker group has been described (see, U.S.Pat. No. 6,048,687); and succinimide esters of Os(phen)₂(dppene)²⁺ andAl(HQS)₃ ³⁺ can be synthesized and attached to nucleic acid sequencesusing similar methods. The Ru(bpy)₃ ²⁺ electrochemiluminescent reportergroup can be synthetically incorporated into nucleic acid sequencesusing commercially available ru-phosphoramidite (IGEN International,Inc., Gaithersburg, Md.).

Additionally other polyaromatic compounds and chelates of ruthenium,osmium, platinum, palladium, and other transition metals have shownelectrochemiluminescent properties. Detailed descriptions of ECL andelectrochemiluminescent moieties can be found in, among other places, A.Bard and L. Faulkner, Electrochemical Methods, John Wiley & Sons (2001);M. Collinson and M. Wightman, Anal. Chem. 65:2576 (1993); D. Brunce andM. Richter, Anal. Chem. 74:3157 (2002); A. Knight, Trends in Anal. Chem.18:47 (1999); B. Muegge et al., Anal. Chem. 75:1102 (2003); H. Abrundaet al., J. Amer. Chem. Soc. 104:2641 (1982); K. Maness et al., J. Amer.Chem. Soc. 118:10609 (1996); M. Collinson and R. Wightman, Science268:1883 et seq. (1995); and U.S. Pat. No. 6,479,233.

The term “reporter probe” refers to a biomolecule, typically anoligonucleotide, that binds to or anneals with at least one(mis)ligation product, at least one (mis)ligation product surrogate, orcombinations thereof, and is used to determine the degree of methylationof at least one target nucleotide. Most reporter probes can becategorized based on their mode of action, for example but not limitedto: nuclease probes, including without limitation TaqMan® probes and thelike (see, e.g., Livak, Genetic Analysis: Biomolecular Engineering14:143-149 (1999); Yeung et al., BioTechniques 36:266-75 (2004));extension probes such as scorpion primers, Lux™ primers, Amplifluors,and the like; hybridization probes such as molecular beacons, Eclipseprobes, and the like; or combinations thereof. Quantitative PCR methods,particularly real-time PCR methods, typically comprise at least onereporter probe, for example but not limited to, at least one nucleaseprobe, at least one hybridization probe, at least one extension probe,at least one probe comprising at least one amide bond, at least oneprobe comprising at least one PNA, at least one probe comprising atleast one LNA, at least one nucleic acid dye, or combinations thereof,including stem-loop and stem-less reporter probes.

In certain embodiments, at least one reporter probe comprises at leastone reporter group, at least one quenching agent, at least one affinitytag, at least one hybridization tag, at least one hybridization tagcomplement, or combinations thereof. In certain embodiments, at leastone hybridization tag complement anneals with at least one hybridizationtag, at least one member of a multi-component reporter group binds to atleast one reporter probe, or combinations thereof. Exemplary reporterprobes include TaqMan® probes; Scorpion probes (also referred to asscorpion primers); Lux primers; FRET primers; Eclipse probes; molecularbeacons, including but not limited to conventional FRET-based molecularbeacons, multicolor molecular beacons, aptamer beacons, PNA beacons,antibody beacons, and probes comprising metallic nanoparticles andsimilar hybrid probes (see, e.g., Dubertret et al., Nature Biotech.19:365-70, 2001). In certain embodiments, such reporter probes furthercomprise groove binders, including but not limited to minor groovebinders, such as but not limited to TaqMan®MGB probes (AppliedBiosystems). In certain embodiments, reporter probes further comprisespanning or bridging oligonucleotides, and enhancer probes, for examplebut not limited to LNA-enhancer probes (see, e.g., Jacobsen et al.,Nucl. Acid Res., 30(19):e100, 2002).

A “substituted hydrocarbon”, as that term is used herein, comprises ahydrocarbon where at least one of the hydrogen atoms in the hydrocarbonassembly is replaced by: a hydrocarbon; a heterocyclic hydrocarbon; asubstituted heterocyclic hydrocarbon; halogen; azide, cyanide,isocyanide, isocyanate, isothiocyanate, —OSO3-, —OSO3R, —SO3-, —SO3R,—OC(O)R, —OC(O)OR, —OR, —CO2R, —C(O)NR2, —NR2, —NRC(O)R, —N(C(O)R)2,—SR, —OP(O)(OR)2, —OP(O)(OR)R, —OP(O)R2, —P(O)(OR)₂, —P(O)(OR)R,—P(O)R2, where R comprises hydrogen, hydrocarbon, heterocyclichydrocarbon, substituted heterocyclic hydrocarbon, or substitutedhydrocarbon. A hydrocarbon comprises an assembly of at least one carbonatoms where any carbon valences not used for forming one or more bondswith another carbon atom are used for bonding with hydrogen atoms. Ahydrocarbon assembly comprises: a linear chain of carbon atoms whereeach of the carbon atoms is connected to a neighboring carbon atom by asingle, double, or triple bond; a cyclic chain of carbon atoms whereeach of the carbon atoms is connected to at least two other carbon atomby a single, double, or in some unusual cases a triple bond; multiplecyclic chains of carbon atoms as described above where at least two ofthe cyclic chains share at least one common carbon-carbon single ormultiple bond to form a fused ring system; multiple cyclic chains ofcarbon atoms as describe above where at least two cyclic chains areconnected together by at least one carbon-carbon single or double bond,but where two bound cyclic chains do not share a common carbon-carbonsingle or double bond.

The term “target nucleic acid sequence” or “target” as used hereinrefers to a specific nucleic acid oligomer, typically genomic DNA, thatcontains one or more target nucleotides. A target nucleotide is thatnucleotide in the target nucleic acid sequence that is interrogated byone or more probes of one or more probe sets to determine itsmethylation state. Generally, a target nucleotide is a cytosine or a5-methylcytosine in a CpG motif, but not always. While the targetnucleic acid sequence is generally described as a single-strandedmolecule, it is to be understood that double-stranded molecules thatcontain one or more target nucleotides are also considered targetnucleic acid sequences. Target nucleic acid sequences can include bothnaturally-occurring and synthetic sequences. The term “template”, whenused in reference to interrogating at least one target nucleotide,typically refers to a synthetic target nucleic acid sequence.

A target nucleic acid sequence according to the present teachings may bederived from any living, or once living, organism, including but notlimited to, prokaryotes, archaea, viruses, and eukaryotes. The targetnucleic acid may originate from the nucleus, typically genomic DNA, ormay be extranuclear, e.g., plasmid, mitochondrial, viral, etc. Theskilled artisan appreciates that genomic DNA includes not only fulllength material, but also fragments generated by any number of means,for example but not limited to, enzyme digestion, sonication, shearforce, and the like. In certain embodiments, the target nucleic acidsequence may be replicated in vitro provided that it retains itsmethylation state, for example without limitation, amplification in thepresence of S-adenyosyl methionine and an appropriate methylase, such asCpG Methylase (M.Sss I) or Human DNA (cytosine-5) Methyltransferase(Dnmt1), commercially available with appropriate reagents from NewEngland Biolabs.

A wide variety of nucleic acid isolation techniques are well known inthe art and are useful in generating target nucleic acid sequences foruse in the teachings herein. Detailed descriptions of such techniquescan be found in, among other places, Ausbel et al.; Rapley; Sambrook etal.; see also, ABI PRISM™ 6100 Nucleic Acid PrepStation and ABI PRISM™6700 Automated Nucleic Acid Workstation (Applied Biosystems, Foster);BloodPrep™ Chemistry and NucPrep™ Chemistry kits (Applied Biosystems).

II. TECHNIQUES

A. Ligation

Ligation according to the present teachings comprises any enzymatic ornon-enzymatic means wherein an inter-nucleotide linkage is formedbetween the opposing ends of nucleic acid probes that are adjacentlyhybridized on a target nucleic acid sequence (i.e., generating a(mis)ligation product). Typically, the opposing ends of the annealednucleic acid probes are suitable for ligation (suitability for ligationis a function of the ligation means employed). In certain embodiments,ligation also comprises at least one gap-filling procedure, wherein theends of the two probes are not adjacently hybridized initially but the3′-end of the upstream probe is extended by one or more nucleotide untilit is adjacent to the 5′-end of the downstream probe, typically by apolymerase (see, e.g., U.S. Pat. No. 6,004,826). The internucleotidelinkage can include, but is not limited to, phosphodiester bondformation. Such bond formation can include, without limitation, thosecreated enzymatically by at least one DNA ligase or at least one RNAligase, for example but not limited to, T4 DNA ligase, T4 RNA ligase,Thermus thermophilus (Tth) ligase, Thermus aquaticus (Taq) DNA ligase,Thermus scotoductus (Tsc) ligase, TS2126 (a thermophilic phage thatinfects Tsc) RNA ligase, Archaeoglobus flugidus (Afu) ligase, Pyrococcusfuriosus (Pfu) ligase, Thermococcus kodakaraensis KOD1 ligaseRhodothermus marinus (Rm) ligase, Methanobacterium thermoautotrophicum(Mth) ligase, Aquifex aeolicus (Aae) ligase, Aeropyrum pemix K1 (Ape)ligase, or the like, including but not limited to, reversiblyinactivated ligases (see, e.g., U.S. Pat. No. 5,773,258), andenzymatically active mutants or variants thereof.

Other internucleotide linkages include, without limitation, covalentbond formation between appropriate reactive groups such as between anα-haloacyl group and a phosphothioate group to form athiophosphorylacetylamino group, a phosphorothioate a tosylate or iodidegroup to form a 5′-phosphorothioester, and pyrophosphate linkages.

Chemical ligation can, under appropriate conditions, occur spontaneouslysuch as by autoligation. Alternatively, “activating” or reducing agentscan be used. Examples of activating and reducing agents include, withoutlimitation, carbodiimide, cyanogen bromide (BrCN), imidazole,1-methylimidazole/carbodiimide/cystamine, N-cyanoimidazole,dithiothreitol (DTT) and ultraviolet light, such as used forphotoligation.

Ligation generally comprises at least one cycle of ligation, i.e., thesequential procedures of: hybridizing the target-specific portions of afirst probe and a corresponding second probe to their respectivecomplementary regions on the corresponding target nucleic acidsequences; ligating the 3′ end of the upstream probe with the 5′ end ofthe downstream probe to form a ligation product; and denaturing thenucleic acid duplex to release the ligation product from the ligationproduct:target nucleic acid sequence duplex. The ligation cycle may ormay not be repeated, for example, without limitation, by thermocyclingthe ligation reaction to amplify the ligation product using ligationprobes (as distinct from using primers and polymerase to generateamplified ligation products). In certain embodiments, ligating orgenerating a (mis)ligation product comprises a multiplicity of cycles ofligation.

Also within the scope of the current teachings are ligation means suchas gap-filling ligation, including, without limitation, gap-filling OLAand LCR, bridging oligonucleotide ligation, and correction ligation.Descriptions of these techniques can be found in, among other places,U.S. Pat. Nos. 5,185,243 and 6,004,826; published European PatentApplications EP 320308 and EP 439182; and PCT Publication Nos. WO90/01069 and WO 01/57268.

A “ligation agent”, according to the present invention, can comprise anynumber of enzymatic or non-enzymatic reagents. For example, ligase is anenzymatic ligation reagent that, under appropriate conditions, formsphosphodiester bonds between the 3′-OH and the 5′-phosphate of adjacentnucleotides in DNA molecules, RNA molecules, or hybrids (depending onthe ligase). Temperature sensitive ligases, include, but are not limitedto, bacteriophage T4 ligase and E. coli ligase. Thermostable ligasesinclude, but are not limited to, Afu ligase, Tag ligase, Tfl ligase, Mthligase, Tth ligase, Tth HB8 ligase, Thermus species AK16D ligase, Apeligase, Lig_(Tk) ligase Aae ligase, Rm ligase, and Pfu ligase (see,e.g., Housby et al., Nucl. Acids Res. 28:e10, 2000; Tong et al., Nucl.Acids Res. 28:1447-54, 2000; Nakatani et al., Eur, J. Biochem.269:650-56, 2002; Zirvi et al., Nucl. Acids Res. 27:e40, 1999; Sriskandaet al., Nucl. Acids Res. 11:2221-28, 2000; and co-filed U.S. ProvisionalPatent Application Ser. No. 60/567,120, filed Apr. 30, 2004, entitled“Compositions, Methods, and Kits for (Mis)ligating Oligonucleotides, byKarger et al.). The skilled artisan will appreciate that any number ofthermostable ligases, including DNA ligases and RNA ligases, can beobtained from thermophilic or hyperthermophilic organisms, for example,certain species of eubacteria and archaea, including viruses that infectsuch thermophilic or hyperthermophilic organisms; and that such ligasescan be employed in the disclosed methods and kits.

Chemical ligation agents include, without limitation, activating,condensing, and reducing agents, such as carbodiimide, cyanogen bromide(BrCN), N-cyanoimidazole, imidazole,1-methylimidazole/carbodiimide/cystamine, dithiothreitol (DTT) andultraviolet light. Autoligation, i.e., spontaneous ligation in theabsence of a ligating agent, is also within the scope of the teachingsherein. Detailed protocols for chemical ligation methods anddescriptions of appropriate reactive groups can be found in, among otherplaces, Xu et al., Nucl. Acids Res., 27:875-81 (1999); Gryaznov andLetsinger, Nucl. Acids Res. 21:1403-08 (1993); Gryaznov et al., NucleicAcid Res. 22:2366-69 (1994); Kanaya and Yanagawa, Biochemistry25:7423-30 (1986); Luebke and Dervan, Nucl. Acids Res. 20:3005-09(1992); Sievers and von Kiedrowski, Nature 369:221-24 (1994); Liu andTaylor, Nucl. Acids Res. 26:3300-04 (1999); Wang and Kool, Nucl. AcidsRes. 22:2326-33 (1994); Purmal et al., Nucl. Acids Res. 20:3713-19(1992); Ashley and Kushlan, Biochemistry 30:2927-33 (1991); Chu andOrgel, Nucl. Acids Res. 16:3671-91 (1988); Sokolova et al., FEBS Letters232:153-55 (1988); Naylor and Gilham, Biochemistry 5:2722-28 (1966);James and Ellington, Chem. & Biol. 4:595-605 (1997); and U.S. Pat. No.5,476,930.

Photoligation using light of an appropriate wavelength as a ligationagent is also within the scope of the teachings. In certain embodiments,photoligation comprises probes comprising nucleotide analogs, includingbut not limited to, 4-thiothymidine (s⁴T), 5-vinyluracil and itsderivatives, or combinations thereof. In certain embodiments, theligation agent comprises: (a) light in the UV-A range (about 320 nm toabout 400 nm), the UV-B range (about 290 nm to about 320 nm), orcombinations thereof, (b) light with a wavelength between about 300 nmand about 375 nm, (c) light with a wavelength of about 360 nm to about370 nm; (d) light with a wavelength of about 364 nm to about 368 nm, or(e) light with a wavelength of about 366 nm. In certain embodiments,photoligation is reversible. Descriptions of photoligation can be foundin, among other places, Fujimoto et al., Nucl. Acid Symp. Ser. 42:39-40(1999); Fujimoto et al., Nucl. Acid Res. Suppl. 1:185-86 (2001);Fujimoto et al., Nucl. Acid Suppl., 2:155-56 (2002); Liu and Taylor,Nucl. Acid Res. 26:3300-04 (1998) and on the world wide web at:sbchem.kyoto-u.ac.jp/saito-lab.

When used in the context of the present teachings, “suitable forligation” refers to at least one first probe and at least onecorresponding second probe, wherein each probe comprises anappropriately reactive group based on the ligation means employed.Exemplary reactive groups include, but are not limited to, a freehydroxyl group on the 3′ end of the upstream probe and a free phosphategroup on the 5′ end of the downstream probe, phosphorothioate andtosylate or iodide, esters and hydrazide, RC(O)S⁻, haloalkyl, RCH₂S andα-haloacyl, thiophosphoryl and bromoacetoamido groups, andS-pivaloyloxymethyl-4-thiothymidine.

B. Amplification

Amplification according to the present invention encompasses any meansby which at least a part of at least one (mis)ligation product, at leastone (mis)ligation product surrogate, or combinations thereof, isreproduced, typically in a template-dependent manner, including withoutlimitation, a broad range of techniques for amplifying nucleic acidsequences, either linearly or exponentially (i.e., generating anamplified (mis)ligation product or generating an amplified digested(mis)ligation product). Exemplary means for performing an amplifyingstep include ligase chain reaction (LCR), PCR, primer extension, stranddisplacement amplification (SDA), multiple displacement amplification(MDA), nucleic acid strand-based amplification (NASBA), rolling circleamplification (RCA), transcription-mediated amplification (TMA), and thelike, including multiplex versions or combinations thereof, for examplebut not limited to, OLA/PCR, PCR/OLA, LDR/PCR, PCR/LDR, LCR/PCR, PCR/LCR(also known as combined chain reaction or “CCR”), and the like.Descriptions of such techniques can be found in, among other places,Sambrook and Russell; Sambrook et al.; Ausbel et al.; PCR Primer: ALaboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); TheElectronic Protocol Book, Chang Bioscience (2002); Msuih et al., J.Clin. Micro. 34:501-07 (1996); Rapley; U.S. Pat. No. 6,027,998; PCTPublication Nos. WO 97/31256 and WO 01/92579; Ehrlich et al., Science252:1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods andApplications, Academic Press (1990); Favis et al., Nature Biotechnology18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000);Belgrader, Barany, and Lubin, Development of a Multiplex LigationDetection Reaction DNA Typing Assay, Sixth International Symposium onHuman Identification, 1995 (available on the world wide web at:promega.com/geneticidproc/ussymp6proc/blegrad.html); LCR Kit InstructionManual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc.Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res.25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999);Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany andGelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Res. 20:1691-96(1992); Polstra et al., BMC Inf. Dis. 2:18-(2002); and Schweitzer andKingsmore, Curr. Opin. Biotechnol. 12:21-7 (2001).

In certain embodiments, amplification comprises at least one cycle ofthe sequential steps of: hybridizing at least one primer withcomplementary or substantially complementary sequences in at least one(mis)ligation product, at least one (mis)ligation product surrogate, orcombinations thereof; synthesizing at least one strand of nucleotides ina template-dependent manner using a polymerase; and denaturing thenewly-formed nucleic acid duplex to separate the strands. The cycle mayor may not be repeated. Amplification can comprise thermocycling or canbe performed isothermally. In certain embodiments, newly-formed nucleicacid duplexes are not initially denatured, but are used in theirdouble-stranded form in one or more subsequent steps and either or bothstrands can, but need not, serve as (mis)ligation product surrogates. Incertain embodiments, single-stranded amplicons are generated and can,but need not, serve as (mis)ligation product surrogates.

Primer extension is an amplifying technique that comprises elongating atleast one probe or at least one primer that is annealed to a template inthe 5′=>3′ direction using an amplifying means such as a polymerase.According to certain embodiments, with appropriate buffers, salts, pH,temperature, and nucleotide triphosphates, including analogs thereof,i.e., under appropriate conditions, a polymerase incorporatesnucleotides complementary to the template strand starting at the 3′-endof an annealed probe or primer, to generate a complementary strand. Incertain embodiments, primer extension can be used to fill a gap betweentwo probes of a probe set that are hybridized to target sequences of atleast one target nucleic acid sequence so that the two probes can beligated together. In certain embodiments, the polymerase used for primerextension lacks or substantially lacks 5′-exonuclease activity.

The term “quantitative PCR”, or “Q-PCR” refers to a variety of methodsused to quantify the results of the polymerase chain reaction forspecific nucleic acid sequences. Such methods typically are categorizedas kinetics-based systems, that generally determine or compare theamplification factor, such as determining the threshold cycle (C_(t)),or as co-amplification methods, that generally compare the amount ofproduct generated from simultaneous amplification of target and standardtemplates. Many Q-PCR techniques comprise reporter probes, intercalatingdyes, or both. For example but not limited to TaqMan® probes (AppliedBiosystems), probes, molecular beacons, Eclipse probes, scorpionprimers, Lux™ primers, FRET primers, ethidium bromide, SYBR® Green I(Molecular Probes), and PicoGreen® (Molecular Probes).

C. Separation

Separating comprises any process that removes at least some unreactedcomponents, at least some reagents, or both some unreacted componentsand some reagents from at least one (mis)ligation product, at least one(mis)ligation product surrogate, or combinations thereof. In certainembodiments, at least one (mis)ligation product, at least one amplified(mis)ligation product, at least one digested (mis)ligation product, atleast one digested amplified (mis)ligation product, or combinationsthereof, are separated from unreacted components and reagents, includingbut not limited to unreacted molecular species present in the sample,ligation reagents, amplification reagents, for example, but not limitedto, unbound/unhybridized ligation probes, primers, enzymes, co-factors,unbound sample components, nucleotides, and the like. The skilledartisan will appreciate that a number of well-known separation means canbe used in the methods disclosed herein.

Exemplary means/techniques for performing a separation step include gelelectrophoresis, including but not limited to isoelectric focusing andcapillary electrophoresis; dielectrophoresis; sorting, including but notlimited to fluorescence-activated sorting techniques; chromatography,including but not limited to HPLC, FPLC, size exclusion (gel filtration)chromatography, affinity chromatography, ion exchange chromatography,hydrophobic interaction chromatography, immunoaffinity chromatography,and reverse phase chromatography; affinity tag binding, such asbiotin-avidin, biotin-streptavidin, maltose-maltose binding protein(MBP), and calcium-calcium binding peptide; aptamer-target binding;hybridization tag-hybridization tag complement annealing; and the like.In certain embodiments, at least one (mis)ligation product, at least one(mis)ligation product surrogate, or combinations thereof are bound toone or more substrates and separated from unbound components. Detaileddiscussion of separation techniques can be found in, among other places,Rapley; Sambrook et al.; Sambrook and Russell; Ausbel et al.; MolecularProbes Handbook; Pierce Applications Handbook; CapillaryElectrophoresis: Theory and Practice, P. Grossman and J. Colburn, eds.,Academic Press (1992); PCT Publication No. WO 01/92579; and M. Ladisch,Bioseparations Engineering: Principles, Practice, and Economics, JohnWiley & Sons (2001).

In certain embodiments, at least one separating step comprises at leastone mobility-dependent analytical technique, for example but not limitedto capillary electrophoresis. In certain embodiments, at least oneseparating step comprises at least one substrate, for example but notlimited to binding at least one biotinylated nucleic acid molecule to atleast one streptavidin-coated substrate. Suitable substrates include butare not limited to microarrays, appropriately treated or coated reactionvessels and surfaces, beads, for example but not limited to magneticbeads, latex beads, metallic beads, polymer beads, microbeads, and thelike (see, e.g., Tong et al., Nat. Biotech. 19:756-59 (2001); Gerry etal., J. Mol. Biol. 292:251-62 (1999); Srisawat et al., Nucl. Acids Res.29:e4 (2001); Han et al., Nat. Biotech. 19:631-35, 2001; and Stears etal., Nat. Med. 9:140-45, including supplements, 2003). Those in the artwill appreciate that the shape and composition of the substrate isgenerally not limiting. In certain embodiments, a plurality of(mis)ligation products, (mis)ligation product surrogates, orcombinations thereof are resolved via a mobility-dependent analyticaltechnique.

In certain embodiments, at least one (mis)ligation product, at least one(mis)ligation product surrogate, or combinations thereof are resolved(separated) by liquid chromatography. Exemplary stationary phasechromatography media for use in the teachings herein includereversed-phase media (e.g., C-18 or C-8 solid phases), ion-exchangemedia (particularly anion-exchange media), and hydrophobic interactionmedia. In certain embodiments, at least one (mis)ligation product, atleast one (mis)ligation product surrogate, or combinations thereof canbe separated by micellar electrokinetic capillary chromatography (MECC).

Reversed-phase chromatography is carried out using an isocratic, or moretypically, a linear, curved, or stepped solvent gradient, wherein thelevel of a nonpolar solvent such as acetonitrile or isopropanol inaqueous solvent is increased during a chromatographic run, causinganalytes to elute sequentially according to affinity of each analyte forthe solid phase. For separating polynucleotides, including (mis)ligationproducts and at least some (mis)ligation product surrogates, anion-pairing agent (e.g., a tetra-alkylammonium) is typically included inthe solvent to mask the charge of phosphate.

The mobility of (mis)ligation products and at least some (mis)ligationproduct surrogates can be varied by using mobility modifiers comprisingpolymer chains that alter the affinity of the probe for the solid, orstationary phase. Thus, with reversed phase chromatography, an increasedaffinity of the (mis)ligation products and at least some (mis)ligationproduct surrogates for the stationary phase can be attained by adding amoderately hydrophobic tail (e.g., PEO-containing polymers, shortpolypeptides, and the like) to the mobility modifier. Longer tailsimpart greater affinity for the solid phase, and thus require highernon-polar solvent concentration for the (mis)ligation products and/or(mis)ligation product surrogates to be eluted (and a longer elutiontime).

In certain embodiments, at least one (mis)ligation product, at least one(mis)ligation product surrogate, or combinations thereof are resolved byelectrophoresis in a sieving or non-sieving matrix. In certainembodiments, the electrophoretic separation is carried out in acapillary tube by capillary electrophoresis (see, e.g., CapillaryElectrophoresis: Theory and Practice, Grossman and Colburn eds.,Academic Press (1992)). Exemplary sieving matrices for use in thedisclosed teachings include covalently crosslinked matrices, such aspolyacrylamide covalently crosslinked with bis-acrylamide; gel matricesformed with linear polymers (see, e.g., U.S. Pat. No. 5,552,028); andgel-free sieving media (see, e.g., U.S. Pat. No. 5,624,800; Hubert andSlater, Electrophoresis, 16: 2137-2142 (1995); Mayer et al., AnalyticalChemistry, 66(10): 1777-1780 (1994)). The electrophoresis medium maycontain a nucleic acid denaturant, such as 7M formamide, for maintainingpolynucleotides in single stranded form. Suitable capillaryelectrophoresis instrumentation are commercially available, e.g., theABI PRISM™ Genetic Analyzer series (Applied Biosystems).

In certain embodiments, at least one hybridization tag complementincludes at least one hybridization enhancer, where, as used herein, theterm “hybridization enhancer” means moieties that serve to enhance,stabilize, or otherwise positively influence hybridization between twopolynucleotides, e.g. intercalators (see, e.g., U.S. Pat. No.4,835,263), minor-groove binders (see, e.g., U.S. Pat. No. 5,801,155),and cross-linking functional groups. The hybridization enhancer may beattached to any portion of a mobility modifier, so long as it isattached to the mobility modifier is such a way as to allow interactionwith the hybridization tag-hybridization tag complement duplex. Incertain embodiments, at least one hybridization enhancer comprises atleast one minor-groove binder, e.g., netropsin, distamycin, and thelike.

The skilled artisan will appreciate that at least one (mis)ligationproduct, at least one (mis)ligation product surrogate, or combinationsthereof can also be separated based on molecular weight and length ormobility by, for example, but without limitation, gel filtration, massspectroscopy, or HPLC, and detected using appropriate methods. Incertain embodiments, at least one (mis)ligation product, at least one(mis)ligation product surrogate, or combinations thereof are separatedusing at least one of the following forces: gravity, electrical,centrifugal, hydraulic, pneumatic, or magnetism.

In certain embodiments, at least one affinity tag is used to separatethe element to which it is bound, e.g., at least one (mis)ligationproduct, at least one (mis)ligation product surrogate, or combinationsthereof, from at least one component of a ligation reaction composition,a digestion reaction composition, an amplified ligation reactioncomposition, or the like. In certain embodiments, at least one affinitytag is used to bind at least one (mis)ligation product, at least one(mis)ligation product surrogate, or combinations thereof to at least onesubstrate, for example but not limited to at least one biotinylated(mis)ligation product, at least one biotinylated (mis)ligation productsurrogate, or combinations thereof, to at least one substrate comprisingstreptavidin. In certain embodiments, at least one aptamer is used tobind at least one (mis)ligation product, at least one (mis)ligationproduct surrogate, or combinations thereof, to at least one substrate(see, e.g., Srisawat and Engelke, RNA 7:632-641 (2001); Holeman et al.,Fold Des. 3:423-31 (1998); Srisawat et al., Nucl. Acid Res. 29(2):e4,2001).

In certain embodiments, at least one hybridization tag, at least onehybridization tag complement, or at least one hybridization tag and atleast one hybridization tag complement, is used to separate the elementto which it is bound from at least one component of a ligation reactioncomposition, a digestion reaction composition, an amplified ligationreaction composition, or the like. In certain embodiments, hybridizationtags are used to attach at least one (mis)ligation product, at least one(mis)ligation product surrogate, or combinations thereof, to at leastone substrate. In certain embodiments, at least one (mis)ligationproduct, at least one (mis)ligation product surrogate, or combinationsthereof, comprise the same hybridization tag. For example but notlimited to, separating a multiplicity of different element:hybridizationtag species using the same hybridization tag complement, tethering amultiplicity of different element:hybridization tag species to asubstrate comprising the same hybridization tag complement, or both.

D. Determining

Determining comprises any means by which the methylation state of one ormore target nucleotide is identified or inferred, including but notlimited to evaluating the degree of methylation of one or more targetnucleotides. In certain embodiments, determining comprises detecting atleast one (mis)ligation product, at least one (mis)ligation productsurrogate, or combinations thereof. In certain embodiments, determiningfurther comprises quantifying the at least one detected (mis)ligationproduct, the at least one detected (mis)ligation product surrogate, orcombinations thereof, for example but not limited to graphicallydisplaying the quantified at least one (mis)ligation product, at leastone (mis)ligation product surrogate, or combinations thereof on a graph,monitor, electronic screen, magnetic media, scanner print-out, or othertwo- or three-dimensional display. Typically the peak height, the areaunder the peak, the signal intensity of one or more detected reportergroup on the (mis)ligation product or (mis)ligation product surrogate,or other quantifiable parameter of the (mis)ligation product orsurrogate are measured and the amount of (mis)ligation product that wasproduced in a particular ligation assay is inferred. Generally, at leastone quantified parameter for at least one (mis)ligation product, atleast one (mis)ligation product surrogate, or combinations thereof, iscompared to the same parameter(s) from a second (mis)ligation product, asecond (mis)ligation product surrogate, or combinations thereof, forexample but not limited to, a competing (mis)ligation product, and aratio of the two (mis)ligation products is obtained.

By comparing the (mis)ligation product ratio obtained from an unknownsample with control ratios or standard curves for the same targetnucleotide and using the same probes and assay conditions, one candetermine the methylation state of the target nucleotide. For example,consider an illustrative competing misligation assay with two possible(mis)ligation products, e.g., LP1 and LP2. Assume in this illustrationthat the LP1:LP2 ratio for a particular unknown sample is 5:1 and theLP1:LP2 ratio obtained using a control target nucleic acid sequenceknown to be fully methylated was 5:1 and with a control target nucleicacid sequence known to be non-methylated was 1:1. By comparing the(mis)ligation product ratio obtained using the unknown sample with thetwo control samples, one can determine that the target nucleotide in theunknown sample was fully methylated. When the ligation product ratioobtained using the unknown sample is between 5:1 and 1:1 in thisexample, one can infer that the degree of target nucleotide methylationhas an intermediate value that depends on those two control ratios.Using the standard curve for that probe set and assay conditions, onecan plot the experimentally determined ligation product ratio on thecurve and determine the corresponding degree of methylation.

In certain embodiments, at least one determining step comprisesdetecting and quantifying at least one (mis)ligation product parameterusing at least one instrument, i.e., using an automated orsemi-automated determining means that can, but need not, comprise acomputer algorithm. In certain embodiments, the determining step iscombined with or is a continuation of at least one separating step, forexample but not limited to a capillary electrophoresis instrumentcomprising at least one fluorescent scanner and at least one graphing,recording, or readout component; a chromatography column coupled with anabsorbance monitor or fluorescence scanner and a graph recorder; or amicroarray with a data recording device such as a CCD camera. Exemplarymeans for performing a determining step include the ABI PRISM® 3100Genetic Analyzer, ABI PRISM® 3100-Avant Genetic Analyzer, ABI PRISM®3700 DNA Analyzer, ABI PRISM® 3730 DNA Analyzer, ABI PRISM® 3730xl DNAAnalyzer (all from Applied Biosystems); the ABI PRISM® 7300 Real-TimePCR System; and microarrays and related software such as the ABI PRISM®1700 (Applied Biosystems) and other commercially available array systemsavailable from Affymetrix, Agilent, and Amersham Biosciences, amongothers (see also Gerry et al., J. Mol. Biol. 292:251-62, 1999; De Belliset al., Minerva Biotec 14:247-52, 2002; and Stears et al., Nat. Med.9:140-45, including supplements, 2003). Exemplary software includesGeneMapper™ Software, GeneScan® Analysis Software, and Genotyper®software (all from Applied Biosystems).

The generation and use of standard curves is well known to those in theart (see, e.g., Overholtzer et al., Proc. Natl. Acad. Sci. 100:11547-52,2003). Typically, a standard curve is generated by plottingexperimentally obtained results for a particular set of reagents andunder defined assay conditions on an X-Y graph or other coordinatesystem and then generating a curve, generally either manually or usingone or more mathematical formula or algorithm, for example but notlimited to graphing and/or line drawing software, linear regressionanalysis and similar mathematical calculations, computer algorithms, orthe like. Once a standard curve have been generated for a given targetnucleotide and at least one corresponding probe set or at least anappropriate subset of at least one corresponding probe set,experimentally-determined results obtained from test (unknown) samplesusing the same probes under the same assay conditions can be evaluatedusing the standard curve and the degree of target nucleotide methylationdetermined. The skilled artisan will appreciate that a “curve” canactually be a straight or substantially straight line or it can becurvilinear and assume a wide range of shapes.

To generate a standard curve for determining the degree of targetnucleotide methylation, (mis)ligation assays are performed under set(“standard”) conditions using appropriate probes, but with at least twotarget compositions comprising different known amounts of methylatedtarget nucleotide sequences. For example but not limited to, a threesample assay where a first ligation reaction composition comprisesnon-methylated target nucleic acid sequences (0% target nucleic acidmethylation), a second ligation reaction composition comprises a 1:1mixture of methylated:non-methylated target nucleotide sequences (50%target nucleotide methylation), and the third ligation reactioncomposition comprises methylated target nucleic acid sequences (100%target nucleotide methylation) and a three point standard curve, usingthe ligation product ratios corresponding to 0, 50 and 100% targetnucleic acid methylation, is generated; a four sample assay where afirst ligation reaction composition comprises non-methylated targetnucleic acid sequences (0% target nucleic acid methylation), a secondligation reaction composition comprises a 1:2 mixture ofmethylated:non-methylated target nucleotide sequences (33.3% targetnucleotide methylation), a third product reaction composition comprisesa 2:1 mixture of methylated:non-methylated target nucleotide sequences(66.6% target nucleotide methylation) and the fourth ligation reactioncomposition comprises methylated target nucleic acid sequences (100%target nucleotide methylation) a four point standard curve, based on theligation product ratios corresponding to 33.3, 50 and 100% targetnucleic acid methylation, is generated; and so forth. The skilledartisan appreciates that the accuracy of standard curves generallyincreases as the number of data points used to generate the curveincreases and also as the number of replicate assays are performed. Theskilled artisan also appreciates that controls and/or calibrationstandards can be included either with unknowns or run in parallel.

According to the present teachings, at least one step for interrogatingat least one target nucleotide is performed using the disclosed probesand probe sets; at least one step for generating at least one(mis)ligation product is performed using the disclosed ligation agentsand ligation techniques; at least one step for generating at least oneamplified (mis)ligation product and/or (mis)ligation product surrogateis performed using the disclosed amplifying means and amplificationtechniques; at least one step for generating at least one digested(mis)ligation product is performed using the disclosed nucleases,restriction enzymes, chemical digesting means, and digestion techniques;and at least one step for determining the degree of methylation of atleast one target nucleotide is performed using at least one discloseddetecting technique, at least one quantifying technique, at least onedisclosed separating technique, or combinations thereof.

Aspects of the present teachings may be further understood in light ofthe following examples, which should not be construed as limiting thescope of the teachings in any way.

III. EXEMPLARY EMBODIMENTS

The present teachings are directed to methods, reagents, and kits thatare useful for determining the degree of target nucleotide methylation.The skilled artisan will appreciate that when analyzing genomic DNAthere are typically multiple copies of the same nucleic acid sequence inthe sample being evaluated, each containing the target nucleotide. Thedegree of methylation for that target nucleotide is generally determinedfrom the sum of at least some of the (mis)ligation products obtainedusing at least part of that population of target nucleic acid sequences.

In certain embodiments, for each target nucleotide to be interrogated,there are at least two probe sets, a first probe set and at least onesecond probe set. In certain embodiments, when the upstream anddownstream probes of the first probe set are hybridized with the targetnucleic acid sequence, the first probe set ligation site includes thecomplement of the target nucleotide. The ligation site for the secondprobe set(s) is a few nucleotides upstream or downstream from the targetnucleotide, as shown in FIG. 1. The first probe set and at least onesecond probe set compete with one another to hybridize with the targetnucleic acid sequence and be ligated. The ligation rate of the firstprobe set compared to the second probe set, i.e., the ligation rateratios, can differ depending on whether the target nucleotide ismethylated.

In certain embodiments, the degree of target nucleotide methylation isdetermined by comparing one or more quantified parameters between two ormore (mis)ligation products or their surrogates, at least one quantified(mis)ligation product parameter and one or more standard curve, or both.In certain embodiments, at least one probe set comprises one or morenucleotides on or near the 3′-end of the upstream probe, on or near the5′-end of the downstream probe, or both, that is not complementary tothe corresponding nucleotide(s) on the target nucleic acid sequence. Thecorresponding nucleotide on the target nucleic acid sequence can, butneed not, be the target nucleotide. In certain embodiments, the ligationsite (in these embodiments, where the misligation occurs), comprises thenucleotide opposing the target nucleotide, as shown in FIG. 2. Incertain embodiments, the ligation site is upstream or downstream of thetarget nucleotide and can (as shown in FIG. 3), but need not, compriseone or more mismatched nucleotide. Those in the art will appreciate thatthe terms upstream or 5′ probe and downstream or 3′ probe are used inreference to their annealing position on the corresponding targetnucleic acid sequence in the 3′=>5′ orientation.

In certain embodiments, at least one ligation rate, at least onemisligation rate, or combinations thereof are changed by the presence ofat least one Modification in at least one probe set. In certainembodiments, at least one ligation rate, at least one misligation rate,or combinations thereof are changed due to changing the hybridizationand or ligation reaction composition or conditions, for example but notlimited to, salt concentration, temperature, changes in one or morecofactor (e.g., α-thio ATP, γ-thio ATP), addition of one or moredenaturant, or the like. In certain embodiments, changing the divalentcation, for example without limitation, substituting a manganese orcalcium salt for a magnesium salt, changes at least one ligation rate,at least one misligation rate, or combinations thereof (see, e.g., Tonget al., Nucl. Acids Res. 28:1447-54, 2000; Nakatani et al., Eur. J.Biochem. 269:650-56, 2002; Tong et al., Nucl. Acids Res. 27:788-94,1999). In certain embodiments, changing at least one ligation rate, atleast one misligation rate, or combinations thereof also changes atleast one ligation rate ratio, at least one misligation rate ratio, orcombinations thereof.

Example 1 Ligation Assay

The degree of target nucleotide methylation was determined using amethylated (comprising a 5-^(Me)C) or non-methylated synthetic modeltemplate: TTATTATGTGGGGCGGACCGCGTGCGCTTACTTAT (SEQ ID NO:1). Theunderlined cytosine is the methylated/non-methylated target nucleotidein this exemplary target nucleic acid sequence. The probe sets used areshown in Table 1. The underlined nucleotide in each probe set isdesigned to be the hybridization partner of the target nucleotide. Theupstream probes in each probe set comprised the fluorescent reportergroup FAM®. The 5′-end of all of the downstream (3′-) probes in this andall other examples described herein were phosphorylated to render themsuitable for ligation. Each assay in this example was performed with atleast two competing probe sets.

TABLE 1 Ligation Probe Sets Probe  Set upstream probe downstream probe 1FAM-AGCGCACGCG GTCCGCCCCAC (SEQ ID NO: 2) probe 2 (SEQ ID NO: 3) probe 32 FAM-AGCGCACGCGGT CCGCCCCACAT (SEQ ID NO: 4) probe 4(SEQ ID NO: 5) probe 5 3 FAM-AGCGCACGCGGTC CGCCCCACATA(SEQ ID NO: 6) probe 6 (SEQ ID NO: 7) probe 7

In this exemplary embodiment, ligation reaction compositions were formedby combining either the methylated or non-methylated synthetic modeltemplate with 12.5 nM of each probe from two of the probe sets shown inTable 1, less than 12.5 nM template, 2 or 4 units of Afu ligase, andligase buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mMdithiothreitol (DTT), 1 mM ATP, and 25 μg/ml bovine serum albumin) in afinal volume of 20 μl. To generate ligation products, the ligationreaction composition was cycled at (65° C. for 5 seconds and 45° C. for1 minute) for 50 cycles, heated to 99° C. for 10 minutes, then cooled to4° C.

Two μL of the ligation product composition was combined with 18 μL Hi-Diformamide (Applied Biosystems) and the diluted ligation products wereseparated and detected using capillary electrophoresis in 36 cmcapillaries with POP-6™ polymer on the ABI PRISM® 3100 Genetic Analyzerin the gene scan mode using GeneScan® Analysis Software according to themanufacturer's instructions (Applied Biosystems). The softwaredetermines, among other things, peak height and peak area (integratedarea under the peak). As shown in FIG. 4, the peak height for theligation product of probes 2 and 3 (“1”) was two to three times higherwith the methylated template than with the non-methylated template,indicating that the ligation rate for Probe Set 1 was enhanced when thetarget nucleotide was methylated. The ligation rates for the other twoprobe sets in this example were much less effected by the methylationstate of the target nucleotide. The ligation product ratios for probeset 1:probe set 2 (“1”/“2”) was 0.6 with the synthetic model templatecomprising the non-methylated target nucleotide and 1.22 with thesynthetic template comprising the methylated target nucleotide; and forprobe set 1:probe set 3 (“1”/“3”), 0.36 with the non-methylated templateand 0.81 with the methylated template (see FIG. 4).

The skilled artisan will appreciate that not every probe or every probeset will satisfactory distinguish the methylated target nucleotide fromthe non-methylated target nucleotide. The skilled artisan understands,however, that appropriate probes and probe sets can be obtained byroutine evaluation of candidate probes and probe sets, without undueexperimentation. Additionally, when using an NAD⁺-dependent ligase,those in the art will understand that NAD+ is generally used as theco-factor in the ligation buffer, rather than ATP. Typically,eubacterial ligases are NAD⁺-dependent while eukaryotic, viral, andarchaeal ligases are ATP-dependent (see, e.g., Weller and Dohertry, FEBSLetters 505:340-342, 2002).

Example 2 Competing Misligation Assay

A probe set comprising a single base mismatch at the 3′ end of each ofthe upstream probes was prepared for interrogating the target nucleotidein the methylated or non-methylated synthetic template corresponding toa segment of the promoter of the P16 tumor suppressor gene:CCAGAGGGTGGGGCGGACCGAGTGCGCTCGGCGGCT (SEQ ID NO:17), where theunderlined “C” is either cytosine (non-methylated template) or5-methylcytosine (methylated template). This probe set comprised threedifferent upstream probes and one downstream probe, shown in Table 2.Each of the upstream probes comprised the fluorescent reporter group FAMand two of the upstream probes comprised polyethylene oxide mobilitymodifiers, shown as (PEO) and (PEO)₂.

TABLE 2 Probe Set 4 5′ probes 3′ probe FAM-AGCGCACTCA(SEQ ID NO: 8) probe 8 FAM-(PEO)-AGCGCACTCC GTCCGCCCCAC(SEQ ID NO: 9) probe 9 (SEQ ID NO: 10) probe 10 FAM-(PEO)₂-AGCGCACTCT(SEQ ID NO: 11) probe 11

In each assay, two competing upstream probes and the downstream probewere used. The ligation reaction composition was generally as describedin Example 1 except that 2 units of Afu ligase, 12.5 nM template, andprobes from Probe Set 4 were used for interrogating the targetnucleotide, in a reaction volume of 10 μL. To generate ligationproducts, the ligation reaction composition was heated to 90° C. for 3minutes, thermocycled (90° C. for 10 seconds, 45° C. for 5 minutes) for40 cycles, heated to 99.9° C. for 20 minutes, then cooled to 4° C. Theligation products were diluted in formamide, separated, detected, andanalyzed as described in Example 1. The ligation product ratio for theligation product of probes 8 and 10 compared to the ligation product ofprobes 9 and 10 (LP 8-10/LP 9-10) was 4.38 when the template comprisingthe non-methylated target nucleotide was interrogated and 8.94 when thetemplate comprising the methylated target nucleotide was interrogated(see FIG. 5A). The ligation product ratio for the ligation product ofprobes 11 and 10 (LP 11-10) compared to LP 8-10 (LP 11-10/LP 8-10) was0.16 when the non-methylated template was used and 0.54 when thetemplate was methylated (see FIG. 5B). The ligation product ratio for LP11-10 compared to LP 9-10 was 0.83 when the non-methylated template wasused and 9.82 when the template comprising the methylated targetnucleotide was interrogated (see FIG. 5C).

Example 3 Competing Misligation Assay

A probe set comprising a single base mismatch at the 3′ end of each ofthe upstream probes (shown underlined in Table 3) was prepared forinterrogating the methylated or unmethylated target nucleotide(underlined) in a synthetic template derived from the transcriptionalregulator gene E2F2: TCCGGGATGCACAGTGCAGAGGCGGCCAGAGCAGTGCACAGCG (SEQ IDNO:12). The probe set comprised three different upstream probes and onedownstream probe. Each of the upstream probes comprised a mismatchednucleotide on its 3′ end (shown underlined) and the fluorescent reportergroup FAM and two of the upstream probes comprised polyethylene oxidemobility modifiers, shown as (PEO) and (PEO)₂ in Table 3.

TABLE 3 Probe Set 5 5′ probes 3′ probe FAM-CACTGCTCTGGCCA(SEQ ID NO: 13) probe 13 FAM-(PEO)-CACTGCTCTGGCCC CCTCTGCACTGTGCAT(SEQ ID NO: 14) probe 14 (SEQ ID NO: 16) probe 16FAM-(PEO)₂-CACTGCTCTGGCCT (SEQ ID NO: 15) probe 15

In each assay there were at least two upstream probes competing to bemisligated to the downstream probe. The ligation reaction composition,reaction conditions, separation, detection and methylation analysis weregenerally as described in Example 2, except that the reactioncomposition was cycled for forty cycles between 90° C. for ten secondsand 50° C. for five minutes.

When probes 13 and 14 were used with probe 16 in this competitionmisligation assay, the ligation product ratio for the ligation productof probes 13 and 16 compared to the ligation product for probes 14 and16 (LP 13-16/LP 14-16) was 4.28 using the non-methylated template and12.18 using the methylated template (see FIG. 6A). When probes 13 and 15were competed, the ligation product ratio (LP 13-16/LP 15-16) was 1.33using the non-methylated template and 4.06 using the methylated template(see FIG. 6B). When probes 14 and 15 were competed, the ligation productratio (LP 14-16/LP 15-16) was 0.35 using the non-methylated template and0.45 using the methylated template (see FIG. 6C).

Example 4 Competing Misligation Assay

A probe set comprising a single base mismatch at the 5′ end of each ofthe downstream probes was prepared for interrogating the targetnucleotide in the synthetic methylated or non-methylated E2F2 template,SEQ ID NO:12. The probe set comprised one upstream probe and threedownstream probes. The upstream probe comprised the fluorescent reportergroup FAM® and the target nucleotide complement (shown underlined), eachof the downstream probes comprised a mismatched nucleotide on the 5′-endand polyethylene oxide mobility modifiers, shown as (PEO), (PEO)₂, and(PEO)₃ in Table 4.

TABLE 4 Probe Set 6 5′ probes 3′ probe ACTCTGCACTGTGCAT-(PEO)(SEQ ID NO: 21) probe 21 FAM-CACTGCTCTGGCCG GCTCTGCACTGTGCAT-(PEO)₂(SEQ ID NO: 22) probe 22 (SEQ ID NO: 23) probe 23TCTCTGCACTGTGCAT-(PEO)₃ (SEQ ID NO: 24) probe 24

Three competition misligation assays (CMAs) were performed in parallel.The first CMA (CMA 1) was performed as follows. A ligation reactioncomposition comprising 12.5 nM upstream probe 22, 12.5 nM downstreamprobe 21, 12.5 nM downstream probe 23, 2 units of Afu ligase, and either0.25 nM methylated E2F2 synthetic template or 0.25 nM non-methylatedE2F2 synthetic template was formed in the ligase buffer described inExample 1, in a final volume of 10 μL. This reaction composition washeated to 90° C. for three minutes, then cycled between 90° C. for tenseconds and 50° C. for five minutes, for sixty cycles, heated to 99.9°C. for twenty minutes, then cooled to 4° C. Two microliters of thiscooled ligation product composition were combined with 18 μL Hi-Diformamide (Applied Biosystems) and loaded onto an ABI PRISM® 3100Genetic Analyzer (Applied Biosystems). The remaining reactionconditions, separation, detection and analysis were generally asdescribed in Example 2. As shown in the top panel of FIG. 7A, the peaksdetected for the two misligation products (LP 22-21 and LP 22-23)obtained with the template comprising the non-methylated targetnucleotide are approximately equal, i.e., the misligation product peakratio is about 1:1. However, the parallel assay using templatescomprising methylated target nucleotides (lower panel) resulted in amisligation product peak ratio of approximately 3:1 (LP 22-23:LP 22-21).

The second CMA was performed in parallel, as described for CMA 1, exceptthat the 12.5 nM downstream probe 24 was used in place of 12.5 nMdownstream probe 23 and the two possible misligation products were LP22-21 and 22-24. As shown in FIG. 7B, the LP 22-24 peak was slightlyhigher than the LP 22-21 peak with the non-methylated template (toppanel). However, the misligation product peak height ratio wasapproximately 4.5:1 (LP 22-24:LP 22-21) with the methylated template(bottom panel).

The third CMA (CMA 3) was performed using 10⁷ copies of either themethylated or unmethylated E2F2 synthetic template, 4 units of Afuligase, upstream probe 22, downstream probes 21 and 24, and cyclingconditions of 90° C. for ten seconds, then 50° C. for two and a halfminutes for 120 cycles, heated at 99.9° C. for 20 minutes, then cooledto 4° C. All other parameters were as described for CMA 1. As shown inFIG. 7C, the misligation product 22-21 peak (LP 22-21) was several timeshigher than the misligation product 22-24 peak (LP 22-24) with thetemplate comprising the non-methylated target nucleotide (top panel).With the template comprising the methylated target nucleotide, however,the height of the LP 22-21 peak was essentially unchanged while theheight of the LP 22-24 peak was dramatically higher (bottom panel) andthe ligation product peak ratio was approximately 4:1 (LP 22-24:LP22-21). Therefore, under these conditions, each of the competitivemisligation assays described in this illustrative embodiment can be usedto determine whether the target nucleotide is methylated or not based onthe respective misligation product peak ratios. Further, the methylationstate of this exemplary target nucleotide can also be determined bycomparing the peak height for LP 22-23 or LP 22-24 using the methylatedtemplate with the corresponding peak height obtained using thenon-methylated template.

Example 5 Competitive Misligation Assay Using Modified Probes

A probe set comprising three downstream probes, each with a single basemismatch at the 5′ end (probes 21, 23, and 24), and a Modified upstreamprobe (probe 22*) comprising a 2′-methoxy-cytosine Modification (shownas C* in Table 5) and a FAM reporter group was synthesized forinterrogating the target nucleotide in the synthetic E2F2 template, SEQID NO:12. Probe 22 (shown in Table 4) and probe 22* (shown in Table 5)differ only by the presence (probe 22*) or absence (probe 22) of the2′-methoxy Modification on the penultimate 3′ cytosine residue. Theligation products were separable in mobility dependent analysistechniques based, at least in part, on the complexity of thepolyethylene oxide mobility modifiers on the respective ligationproducts, shown in Table 5 as (PEO), (PEO)₂, and (PEO)₃ on thedownstream probes.

TABLE 5 Probe Set 7 5′ probes 3′ probe ACTCTGCACTGTGCAT-(PEO)(SEQ ID NO: 21) probe 21 FAM-CACTGCTCTGGCC*G GCTCTGCACTGTGCAT-(PEO)₂probe 22* (SEQ ID NO: 23) probe 23 TCTCTGCACTGTGCAT-(PEO)₃(SEQ ID NO: 24) probe 24Each assay included two competing downstream probes and the upstreamprobe. The ligation reaction composition, reaction conditions,separation, detection and analysis were generally as described inExample 4.

When probes 21 and 23 were used with probe 22* in this competitionmisligation assay, the ligation product ratio for the ligation productof probes 22* and 23 compared to the ligation product for probes 22* and23 (LP 22*-23/LP 22*-21) was 1.13 using the synthetic E2F2 templatecomprising the non-methylated target nucleotide and 3.09 using themethylated template (see FIG. 8A). When probes 21 and 24 were used withprobe 22* in this competition misligation assay, the ligation productratio for the ligation product of probes 22* and 24 compared to theligation product for probes 22* and 21 (LP 22*-24/LP 22*-21) was 2.69using the synthetic template comprising the non-methylated targetnucleotide and 7.9 using the methylated template (see FIG. 8B).

Example 6 Competing Misligation Assay with Amplification Using gDNA

To evaluate the competing misligation assay for interrogating the sameE2F2 target nucleotide in gDNA instead of a synthetic oligonucleotide,non-methylated and methylated human gDNA was obtained from publicsources (Coriell Institute for Medical Research, Camden, N.J. andSerologicals Corp. Nocross, Ga., respectively). Due to possible low copynumber of a particular target nucleic acid sequence in gDNA anamplification step was included in this exemplary embodiment. A probeset comprising two upstream probes and three downstream probes wassynthesized, as shown in Table 6. Each of the probes comprised either a“universal” upstream primer-binding portion or a “universal” downstreamprimer-binding portion (shown in brackets) and each the downstreamprobes comprised a mismatched nucleotide on its 5′ end. Probes 27 and 28also included a mobility modifier comprising several non-sequencerelated nucleotides (underlined) to enhance ligation product separation.The target nucleotide complement was on the 3′-end of the upstream probe(underlined). A Modified version of probe 25 (probe 25*) was synthesizedwith a 2-methoxy Modification on the penultimate cytosine (shown as C*).

TABLE 6 Probe Set 8 5′ probes 3′ probe [CTCGTAGACTGCGTACCGATC]CAACTCTGCACTGTGCAT- CTGCTCTGGCCG [TTACTCAGGACTCATCTCGC](SEQ ID NO: 25) probe 25 (SEQ ID NO: 26) probe 26[CTCGTAGACTGCGTACCGATC]CA GCTCTGCACTGTGCATTTTT- CTGCTCTGGCC*G[TTACTCAGGACTCATCGTCGC] probe 25* (SEQ ID NO: 27) probe 27TCTCTGCACTGTGCATTTTT- [TTACTCAGGACTCATCGTCGC] (SEQ ID NO: 28) probe 28Two sets of parallel ligation reaction compositions (four reactioncompositions) were prepared in a final volume of 10 μL as follows: 25nanograms (ng) of either (i) methylated or (ii) unmethylated gDNA targetnucleic acid sequences; 12.5 nM probe 25; 12.5 nM of other either (iii)probe 26 or (iv) probe 27; and 2-4 units of Afu ligase, all in reactionbuffer as described in Example 1. To generate misligation products, theligation reaction compositions were heated to 90° C. for three minutes,cycled one hundred twenty times between 90° C. for ten seconds and 50°C. for two and a half minutes, heated to 99.9° C. for twenty minutes,then cooled to 4° C.

Amplification reaction compositions were formed by separately combiningeach of these ligation product compositions with 0.5 units of Taq Gold™polymerase (Applied Biosystems) and 0.5 μM of each of the universalamplification primers, FAM-CTCGTAGACTGCGTACCGATC (SEQ ID NO:29; FAM:fluorescent reporter group FAM®, Applied Biosystems) andGCGACGATGAGTCCTGAGTAA (SEQ ID NO:30). To generate amplified misligationproducts, the amplification reaction compositions were then heated to95° C. for ten minutes, and cycled between 94° C. for ten seconds and68° C. for one minute for 25-30 cycles, then cooled to 4° C. Two μL ofthe amplified misligation products were diluted with 18 μL Hi-Di™formamide. The diluted amplified misligation products were loaded ontoan ABI PRISM® 3100 Genetic Analyzer and separated and analyzed, asdescribed in Example 1. By comparing the ratio of the amplifiedmisligation product (i.e., one form of misligation product surrogate)peaks shown in FIGS. 9A and 9B (LPS 25-26, LPS 25-27, and LPS 25-28),one can determine the methylation state of the target nucleotide.

The ligation product ratio, based on the peak area of the misligationproduct surrogate for the misligation product of probes 25 and 27 (LPS25-27) compared to the misligation product of probes 25 and 26 (LPS25-26) was 1.72 when the gDNA comprising the non-methylated targetnucleotide was interrogated and 4.37 when the gDNA comprising themethylated target nucleotide was interrogated (see FIG. 9A). Theligation product ratio, based on the peak area of LPS 25-26 compared tothat for the misligation product surrogate for the ligation product ofprobes 25 and 28 (LPS 25-28) was 3.38 when the gDNA comprising thenon-methylated target nucleotide was interrogated and 7.24 when the gDNAcomprising the methylated target nucleotide was interrogated (see FIG.9B).

To evaluate the use of Modified probes for methylation determinationsusing gDNA target nucleic acid sequences, an upstream probe comprising aModification was prepared by adding a 2′-methoxy Modification to thepenultimate nucleotide of probe 25 (see probe 25* in Table 7). Theligation reaction composition was prepared as previously described inthis example except that probe 25* was used in place of probe 25 anddownstream probes 27 and 28 were competed against each other. All otheraspects of the misligation assay and amplification were the same.

As shown in FIG. 9C, Modified probe 25* also affected the misligationrate, allowing the methylation status of the exemplary target nucleotideto be determined. The ligation product ratio, based on the area underthe peak of the misligation product surrogate for the misligationproduct of probes 25* and 28 (LPS 25*-28) compared to that of themisligation product surrogate for the misligation product of probes 25*and 27 (LPS 25*-27) was 1.41 with the gDNA comprising the non-methylatedtarget nucleotide and 2.24 with the gDNA comprising the methylatedtarget nucleotide (see FIG. 9C).

Example 7 Competing Misligation Assay with Amplification Using gDNA

A second competing misligation assay followed by digestion andamplification was performed to determine the methylation status of thesame E2F2 target nucleotide in gDNA as in Example 6. Two ligationreaction compositions were formed as described in Example 6 except thatprobes 31, 32, and 25 were combined in one ligation reaction compositionand probes 31, 33, and 25 were combined in the other. As shown in Table7, probes 31, 32, and 33 each comprise a universal downstreamprimer-binding portion (shown in brackets), one of two hybridizationtags (shown in italics), and a mismatched nucleotide at the 5′-end ofthe probe. Probes 25 and 25* contain a universal upstream primer-bindingportion (shown in brackets) and the target nucleotide complement at the3′-end (underlined).

TABLE 7 Probe Set 9 5′ probe 3′ probes [CTCGTAGACTGCGTACCGATC]ACTCTGCACTGTGCAT- CACTGCTCTGGCCG TCGCAGATTGTGTCTCACCGAGGA- probe 25[TTACTCAGGACTCATCGTCGC] (SEQ ID NO: 31) probe 31 [CTCGTAGACTGCGTACCGATC]GCTCTGCACTGTGCAT- CACTGCTCTGGCC*G CGATTCAAACTGAAGCGTGCCGACG- probe 25*[TTACTCAGGACTCATCGTCGC] (SEQ ID NO: 32) probe 32 TCTCTGCACTGTGCAT-CGATTCAAACTGAAGCGTGCCGACG- [TTACTCAGGACTCATCGTCGC](SEQ ID NO: 33) probe 33

The misligation products were generated as described in Example 6,except downstream probes 31, 32, and 33 were used. Each of thesemisligation product compositions were then digested with exonuclease bycombining five μL of ligation reaction composition with five μL ofexonuclease solution (0.2 μL λ exonuclease (1 Unit; New EnglandBioLabs), 0.5 μL 10×λ exonuclease buffer (New England Biolabs), 4.3 μLdistilled water). To generate digested misligation products, the twodigestion compositions were heated to 37° C. for ninety minutes, thenheated to 80° C. for ten minutes. Each of the digested misligationproduct compositions were diluted by adding 15 μL of distilled water.

Digested amplification reaction compositions were formed by combining2.08 μL of the diluted digested misligation product composition with7.92 μL PCR premix (0.5 Units AmpliTaq Gold™ DNA Polymerase (AppliedBiosystems), 50 nM Tris-HCl, pH 8.0 at 25° C., 2.5 mM MgCl₂, 0.01%sodium azide, 0.01% Tween 20, 8% glycerol (v/v), 0.1 mM deferoxaminemesylate, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dUTP and 0.5 μMeach of biotin-CTCGTAGACTGCGTACCGATC (SEQ ID NO:34) comprising a biotinmoiety at its 5′-end, and GCGACGATGAGTCCTGAGTAA (SEQ ID NO:35)). Togenerate digested amplified misligation products, the digestedamplification reaction compositions were heated to 95° C. for tenminutes, then cycled between 94° C. for ten seconds and 68° C. for oneminute for 25-30 cycles to generate double-stranded amplicons comprisingone biotinylated strand (i.e., a form of misligation product surrogate).

The wells of a streptavidin plate (Roche Bioscience) were washed threetimes with 25 μL Wash Buffer (1/10 dilution of 1×SSC, 0.1% Tween 20).One part of the biotinylated amplicons was diluted in seven partshybridization buffer (1×SSC, 0.01% Tween 20) to form a hybridizationmix. Twenty μL of this hybridization mix was added to wells of thewashed streptavidin plate and incubated at room temperature on anorbital shaker. After a 30 minute incubation, the liquid in each wellwas removed and the wells were washed three times with 30 μL WashBuffer. Fifty μL of 0.1N NaOH was added to the wells and the plate wasincubated at room temperature on an orbital shaker. After five minutes,the wells were emptied then washed five times with 50 μL Wash Buffer.

ZipChute solution was prepared by combining 6.576 mL 7.3× ZipChutedilution buffer, 5.40 mL omnipure formamide, and 0.024 mL of 250 nMZipChute stock solution (Applied Biosystems). Twenty-five μL ZipChutesolution was added to the wells and the plate was incubated at 37° C.After one hour, the wells were emptied, washed four times with 25 μLWash Buffer, then spin dried. Next, 17.5 μL SNPlex loading reagent(Applied Biosystems) was added to the individual wells and the plate wasincubated at 37° C. to release the ZipChutes (i.e., a form of(mis)ligation product surrogate) from the wells of the plate into theloading reagent. After a thirty minute incubation, ten μL of the loadingreagent comprising released ZipChutes from individual wells of thestreptavidin plates were transferred to individual wells of a 384 wellplate. These samples were analyzed on an ABI PRISM® 3100 GeneticAnalyzer, essentially as described above.

As shown in FIG. 10A, the LPS 25-32:LPS 25-31 peak area ratio was 0.95with the non-methylated gDNA and 1.66 with methylated gDNA. The LPS25-33:LPS 25-31 peak area ratio was 2.31 with the non-methylated gDNAand 6.22 with methylated gDNA (see FIG. 10B). The LPS 25*-33:LPS 25*-31peak area ratio was 0.53 with the non-methylated gDNA and 2.49 withmethylated gDNA (see FIG. 10C).

Example 8 Competing Misligation Assay with Amplification Using gDNA

To evaluate the competing misligation assay with the P16 targetnucleotide shown in SEQ ID NO:17 in the context of gDNA, three parallelligation reaction compositions were formed as described in Example 7except that probes 36, 37 and 38 were combined in a first ligationreaction composition, probes 36, 37, and 40 were combined in a secondligation reaction composition, and probes 37, 39, and 40 were combinedin a third ligation reaction composition. As shown in Table 8, probes36, 38, 39, and 40 each comprise a universal upstream primer-bindingportion (shown in brackets), one of two hybridization tags (shown initalics), and a mismatched nucleotide at the 3′-end of the probe. Probe37 contains a universal downstream primer-binding portion (shown inbrackets) and the target nucleotide complement at its 5′-end(underlined).

TABLE 8 Probe Set 10 5′ probes 3′ probe [CTCGTAGACTGCGTACCGATC]TCCTCGGGTCCGCCCCAC[TTACT TGAGACACAATCTGCGAAGCGCACTCA CAGGACTCATCGTCGC](SEQ ID NO: 36) probe 36 (SEQ ID NO: 37)  probe 37[CTCGTAGACTGCGTACCGATC]CGTCGGC ACGCTTCAGTTTGAATCGAGCGCACTCC(SEQ ID NO: 38) probe 38 [CTCGTAGACTGCGTACCGATC]TCCTCGGTGAGACACAATCTGCGAAGCGCACTCC (SEQ ID NO: 39) probe 39[CTCGTAGACTGCGTACCGATC]CGTCGGC ACGCTTCAGTTTGAATCGAGCGCACTCT(SEQ ID NO: 20) probe 40The remainder of the misligation assay, digestion, amplification,separation, detection and determination were performed as described inExample 6, except that the primers used werebiotin-GCGACGATGAGTCCTGAGTAA (SEQ ID NO:18) and CTCGTAGACTGCGTACCGATC(SEQ ID NO:19). As shown in FIG. 11A, the digested amplified misligationproduct (i.e., a form of misligation product surrogate) peak heightratios obtained from the first ligation product reaction composition(LPS 36-37:LPS 38-37) shows little to no change between the methylatedand non-methylated target. As shown in FIG. 11B, the ligation productsurrogate peak height ratio obtained from the second ligation reactioncomposition for the non-methylated template is approximately 3:4 (LPS36-37:LPS 40-37), but shifts to 4:2 (LPS 36-37:LPS 40-37) with themethylated gDNA. The misligation product surrogate peak height ratiosfor the third ligation reaction composition also varied between thenon-methylated and methylated gDNA, as shown in FIG. 11C. With thenon-methylated gDNA (upper panel), the ligation product surrogate peakheight ratio was approximately 1:3 (LPS 39-37:LPS 40-37), while it wasapproximately 3:2 (LPS 39-37:LPS 40-37) with the methylated gDNA (lowerpanel). Thus, under these conditions, the competing probes used in thesecond and third of these misligation assays are useful in determiningthe methylation of the illustrative P16 target nucleotide in gDNA whilethose used in the first reaction composition of this example were lesseffective. As the person in the art appreciates, identification ofuseful probes and probe sets can be determined through routineevaluation using the disclosed teachings and without undueexperimentation.

Example 9 Generating a Standard Curve

One way to determine the degree of target nucleotide methylation is tocompare the experimental results obtained according to the presentteachings with a corresponding standard curve. A standard curve can begenerated by combining at least one upstream probe and at least onecorresponding downstream probe from a probe set with a target comprisinga pre-determined mixture of methylated and non-methylated target nucleicacid sequences. For example, for each of the ligation reactioncompositions of Example 8, six parallel compositions are prepared withthe gDNA target comprising: (i) 25 ng methylated gDNA, (ii) 20 ngmethylated gDNA and 5 ng non-methylated gDNA, (iii) 15 ng methylatedgDNA and 10 ng non-methylated gDNA, (iv) 10 ng methylated gDNA and 15 ngnon-methylated gDNA, (v) 5 ng methylated gDNA and 20 ng non-methylatedgDNA, or (vi) 25 ng non-methylated gDNA, respectively. The remainder ofthe reaction conditions and techniques are as described in Example 8.

For each of the possible (mis)ligation products in each set of ligationreaction compositions, e.g., LP 36-37 and LP 38-37, there are six(mis)ligation product peak height ratios corresponding to 0, 20, 40, 60,80 and 100% methylated target (or vice versa). A plot of, for example,percent methylation versus (mis)ligation product peak ratio is generatedand the data points fit to a curve, i.e., a “standard curve” for theprobes tested. Using this standard curve, one can determine the degreeof target nucleotide methylation in an unknown sample by locating theexperimentally determined (mis)ligation product peak ratio at theappropriate point on the curve and identifying the corresponding degreeof methylation, provided that the same probes and assay conditions areused for creating the standard curve and obtaining the unknown sample'sligation product ratio. Those skilled in the art understand that thereliability of standard curves is improved by, among other things,increasing the number of data points used to generate the “curve” andthe number of replicates obtained for each data point. Those in the artalso understand that standard curves can be generated using any or anumber of measurable parameters, not just (mis)ligation product peakheight. For example but without limitation, peak height and peak areamay be routinely determined using software such as GeneScan™ orGeneMapper™ software and provided as part of a system printout orgraphic display.

Example 10 Evaluating the Methylation Detection Potential of FourLigases

The methylation detection potential of Afu, AK16D, Taq, and Tth ligaseswere evaluated in a series of ligation assays using probe sets 1, 2, and3 (shown in Table 1) with either the methylated or the unmethylatedsynthetic model template, SEQ ID NO:1. Each 20 μL ligation reactioncomposition comprised 4 Units of ligase (Afu, AK16D, Taq, or Tth), 12.5nM template (either methylated or unmethylated SEQ ID NO:1), and 12.5 nMof each of the six probes from probe sets 1-3 in 1× ligase buffer (forAfu ligase: 50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 10 mM dithiothreitol(DTT), 1 mM ATP, 25 μg/ml bovine serum albumin; for AK16D, Taq, and Tthligases: 20 mM Tris-HCl, pH 7.6, 25 mM potassium acetate, 10 mMmagnesium acetate, 10 mM DTT, 1 mM NAD, 0.1% Triton X-100). Ligationproducts were generated by heating the ligation reaction compositions at85° C. for 3 minutes, cycling twenty-five times at (85° C. for fiveseconds, 40° C. for 2 minutes), heating at 95° C. for ten minutes, thencooled to 4° C. Two μL of the ligation products were diluted in 18 μLHi-Di™ formamide, then the diluted (mis)ligation products were loadedonto capillaries and separated on the ABI PRISM® 3100 Genetic Analyzer,as described.

The detected ligation product peaks obtained with Afu ligase are shownin FIG. 12A. The ligation product ratio with the non-methylated andmethylated template for LP 2-3/LP4-5 was 0.44 (non-methylated) and 0.78(methylated); for LP 2-3/LP 6-7 was 0.32 (non-methylated) and 0.78(methylated); and for LP 4-5/LP 6-7 was 0.72 (non-methylated) and 0.76(methylated). The detected ligation product peaks obtained with AK16Dligase are shown in FIG. 12B. The ligation product ratio with thenon-methylated and methylated template for LP 2-3/LP4-5 was 0.30 and0.41, respectively; for LP 2-3/LP 6-7 was 0.37 and 0.46, respectively;and for LP 4-5/LP 6-7 was 1.24 and 1.12., respectively. The detectedligation product peaks obtained with Tth ligase are shown in FIG. 12C.The ligation product ratio with the non-methylated and methylatedtemplate for LP 2-3/LP4-5 was 0.50 and 0.53, respectively; for LP 2-3/LP6-7 was 0.41 and 0.42, respectively; and for LP 4-5/LP 6-7 was 0.82 and0.79, respectively. The detected ligation product peaks obtained withTaq ligase are shown in FIG. 12D. The ligation product ratio with thenon-methylated and methylated template for LP 2-3/LP4-5 was 0.58 and0.60, respectively; for LP 2-3/LP 6-7 was 0.51 and 0.47, respectively;and for LP 4-5/LP 6-7 was 0.87 and 0.78, respectively. Those in the artwill appreciate that similar evaluations of additional ligases can bepreformed using the same or different templates and/or probes toevaluate the potential of those ligases for detecting methylated targetnucleotides under a given set of experimental conditions.

While the present teachings have been described in terms of theseexemplary embodiments, the skilled artisan will readily understand thatnumerous variations and modifications of these exemplary embodiments arepossible without undue experimentation. All such variations andmodifications are within the scope of the current teachings.

1-69. (canceled)
 70. A kit for determining the degree of methylation ofat least one target nucleotide in at least one target nucleic acidsequence, comprising at least one ligation probe set and at least oneligation agent.
 71. The kit of claim 79, wherein the at least onethermostable ligase comprises Afu ligase.
 72. The kit of claim 70,further comprising: at least one amplifying agent; at least one primer;at least one reporter group; at least one reporter probe; at least onemobility modifier moiety; at least one hybridization tag; at least onehybridization tag complement; or combination thereof.
 73. The kit ofclaim 72, wherein the at least one amplifying agent comprises at leastone thermostable polymerase.
 74. A kit for determining the degree ofmethylation of at least one target nucleotide comprising: at least onemeans for ligating; at least one means for amplifying; at least onemeans for separating; at least one means for digesting; at least onemeans for quantifying, or combination thereof. 75-76. (canceled)
 77. Thekit of claim 70, wherein the at least one ligation agent is a ligase.78. The kit of claim 77, wherein the at least one ligation agent is aDNA or an RNA ligase.
 79. The kit of claim 77, wherein the ligase is atleast one thermostable ligase.
 80. The kit of claim 79, wherein thethermostable ligase is selected from group consisting of Thermus speciesligase, Pfu ligase, and Afu ligase.
 81. The kit of claim 73, wherein theat least one thermostable polymerase is selected from the groupconsisting of Taq polymerase, Tfl polymerase, Tth polymerase, Tlipolymerase, Pfu polymerase, AMPLITAQ GOLD polymerase, 9° N_(m)™polymerase, VENT_(R) DNA polymerase, DEEP VENT_(R) DNA polymerase, andUlTma polymerase.
 82. The kit of claim 74, wherein the thermostablepolymerase is AMPLITAQ GOLD polymerase.